JP7220755B2 - NOVEL ANTI-CD3 ANTIBODY AND USES THEREOF - Google Patents

Description

The present invention relates to anti-CD3 antibodies and fragments thereof conjugated with folic acid.

Naturally produced antibodies (Abs) are tetrameric structures composed of two identical immunoglobulin (Ig) heavy chains and two identical immunoglobulin light chains. The heavy and light chains of Abs consist of distinct domains. Each light chain has one variable domain (VL) and one constant domain (CL). Each heavy chain, on the other hand, has one variable domain (VH) and three or four constant domains (CH). Each domain (consisting of approximately 110 amino acid residues) folds into a characteristic β-sandwich structure (immunoglobulin fold) formed from two antiparallel β-sheets. Each VL domain has three complementarity determining regions (CDR1-3) and each VH domain has up to four complementarity determining regions (CDR1-4). A CDR is a loop (or turn) that joins a β-strand at one end of a variable domain. Both the light and heavy chain variable regions are typically responsible for antigen specificity, although individual chains may not contribute equally to specificity. Antibody molecules have evolved to bind multiple molecules by randomizing the CDR loops.

Functional substructures of Abs can be produced by proteolytic and recombinant methods. Abs include Fab fragments, Fv fragments and Fc portions. The Fab fragment contains the VH-CH1 domain of the heavy chain and the VL-CL1 domain of the light chain, linked by a single interchain disulfide bond. Fv fragments contain only the VH and VL domains. The Fc portion includes regions of the antibody molecule that do not bind antigen. In some instances even a single VH domain retains significant affinity for antigen (Ward et al., 1989, Nature 341, 554-546). Certain monomeric kappa light chains have also been shown to bind specifically to their antigens (L. Masat et al., 1994, PNAS 91:893-896). It has been found that isolated light or heavy chains may also retain some antigen-binding activity (Ward et al., 1989, Nature 341, 554-546).

Other functional substructures include single chain Fv (scFv). The scFV consists of immunoglobulin heavy and light chain variable regions, which are covalently linked by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small proteins (Mr: 25,000) usually retain their specificity and affinity for antigen in the form of one polypeptide. And can provide convenient building blocks for larger antigen-specific molecules. scFv have short circulating half-lives and are often of limited therapeutic utility.

Small protein scaffolds called "minibodies" have been designed using part of the VH domain of Ig as a template (Pessi et al., 1993, Nature 362, 367-369). A minibody with a high affinity for interleukin 6 (dissociation constant (K _d ): ˜10 ⁻⁷ M) randomizes the loops corresponding to CDR1 and CDR2 of VH, and mutants are then selected by phage display. It has been discovered (Martin et al., 1994, EMBO J. 13, 5303-5309).

Analysis of IgG-like material from camel serum usually lacks the light chain variable domain. This suggests that the full specificity and affinity of an antibody can derive from the VH domain (3 or 4 CDR loops) only. High affinity "camelized" VH domains have been generated. Moreover, high specificity can be obtained by randomizing only CDR3.

An alternative to "minibody" is "diabody". A diabody is a small bivalent antibody fragment with bispecificity and has two antigen-binding sites. This fragment contains a heavy chain variable domain (VH) and a light chain variable domain (VL), which are linked on the same polypeptide chain (VH-VL). The size of the diabody is similar to that of the Fab fragment. The diabody linker is too short for pairing between the two domains on the same strand. Each domain must therefore match with the corresponding domain on the other strand. Thus, two antigen binding sites are created. These dimeric antibody fragments (or "diabodies") are bivalent and have bispecificity (see P. Holliger et al., PNAS 90:6444-6448 (1993).). .

CDR peptides and CDR organomimetics have been generated (Dougall et al., 1994, Trends Biotechnol. 12, 372-379). CDR peptides are short peptides, usually cyclic, that correspond to the amino acid sequences of the CDR loops of an antibody. CDR loops are responsible for antibody-antigen interactions. CDR peptides and CDR organomimetics have been shown to retain some binding affinity (Smyth & von Itzstein, 1994, J. Am. Chem. Soc. 116, 2725-2733). Mouse CDRs have been grafted onto human Ig scaffolds without loss of affinity (Jones et al., 1986, Nature 321, 522-525; Riechmann et al., 1988).

In vivo, specific Abs are selected from a large library and amplified (affinity maturation). This process can be reproduced in vitro using combinatorial library technology. Successful display of Ab fragments on the surface of bacteriophage allows the generation and screening of large numbers of CDR variants (McCafferty et al., 1990, Nature 348, 552-554; Barbas et al., 1991 , Proc. Natl. Acad. Sci. USA 88, 7978-7982; Winter et al., 1994, Annu. An increasing number of Fabs and Fvs (and their derivatives) are produced by this technology. Combinatorial techniques can also be combined with pseudo Abs.

A number of protein domains that are potential protein scaffolds have been expressed fused to the phage capsid protein. For a review see Clackson & Wells, Trends Biotechnol. 12:173-184 (1994). Some of these protein domains have already been used as scaffolds to display random peptide sequences. Examples of this include bovine pancreatic trypsin inhibitor (Roberts et al., PNAS 89:2429-2433 (1992)), human growth hormone (Lowman et al., Biochemistry 30:10832-10838 (1991)), Venturini et al. , Protein Peptide Letters 1:70-75 (1994)), the IgG binding domain of Streptococcus (O'Neil et al., Techniques in Protein Chemistry V (Crabb, L,. ed.) pp. 517-524, Academic Press, San Diego (1994)). These scaffolds present one randomized loop or region. Tendamistat has been used as a scaffold for display on the filamentous phage M13 (McConnell and Hoess, 1995, J. Mol. Biol. 250:460-470).

Covalent attachment of the hydrophilic polymer polyethylene glycol (abbreviated PEG) to (i) improved water solubility and bioavailability, (ii) increased serum and therapeutic half-life, and (iii) ) immunogenicity and biological activity are modulated, or (iv) for molecules with many biological activities (eg, proteins, peptides, and particularly hydrophobic molecules), circulation time is increased. PEG has been used extensively in pharmaceuticals, artificial implants, and other applications where biocompatibility, elimination of toxicity and elimination of immunogenicity are important. In order to maximize the desired properties of PEG, the total molecular weight and hydration state of the PEG polymer or polymer attached to the bioactive molecule must be sufficiently high. In this way, the general advantages associated with the addition of PEG polymers can be conferred (increased water solubility, increased half-life in circulation, etc.) without adversely affecting the biological activity of the parent molecule. extension, etc.).

Frequently, PEG derivatives are linked to biologically active molecules via reactive chemically functional moieties such as lysine, cysteine and histidine residues, N-termini, and carbohydrate moieties. Proteins and other molecules typically have only a limited number of reactive sites available for polymer attachment. Generally, the most favorable sites for polymer addition modification play an important role in receptor binding and are essential for retaining biological activity of the molecule. Consequently, the indiscriminate addition of polymer chains to the reactive sites of biologically active molecules usually leads to a significant reduction or complete loss of biological activity of the polymer-modified molecule (R. Clark et al. ., (1996), J. Biol. Chem., 271:21969-21977). Conventional approaches to forming conjugates with sufficient polymer molecular weight to impart a desired benefit to a target molecule have generally involved the random addition of multiple polymer chains to the molecule. Therefore, it has been an approach with a high risk of reducing or completely eliminating the biological activity of the parent molecule.

Amino acids that are not genetically encoded can be incorporated into proteins. This introduces chemical functional groups that can provide a variety of alternatives to natural functional groups (e.g., ε-NH ₂ of lysine, sulfhydryl-SH of cysteine, imino group of histidine, etc.). be able to. The specific chemical functional group is inactive to the functional group of the 20 types of ordinary amino acids (amino acids encoded by genes), but reacts efficiently and efficiently to form a stable bond. is known to form For example, azide and acetylene groups are known to those skilled in the art to undergo Huisgen [3+2] cycloaddition reactions under aqueous conditions and in the presence of catalytic amounts of copper (e.g., Tornoe, et al., ( 2002) Org. Chem. 67:3057-3064; Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599.). By introducing an azide moiety into the protein structure, for example, it is inert to amine groups, sulfhydryl groups, carboxylic acid groups, and hydroxy groups in proteins, while reacting smoothly and efficiently with acetylene moieties. Functional groups can be incorporated such that they form cycloaddition products. Importantly, in the absence of the acetylene moiety, the azide remains chemically inert and unreactive in the presence of other protein side chains and under physiological conditions.

The present invention specifically addresses the problem of activity and production of antigen-binding polypeptides and fragments thereof (particularly polypeptide-small molecule complexes). The invention also addresses the production of antigen-binding polypeptides and polypeptide-small molecule conjugates with enhanced biological or pharmaceutical properties (eg, increased therapeutic half-life).

The present invention provides anti-CD3 antibodies, and conjugates thereof with folic acid. In some embodiments, the novel anti-CD3 antibodies of the invention comprise one or more non-naturally encoded amino acids. In some embodiments, the anti-CD3 antibody comprises a complete antibody heavy chain. In some embodiments, the anti-CD3 antibody comprises a complete antibody light chain. In some embodiments, the anti-CD3 antibody comprises the variable region of an antibody light chain. In some embodiments, the anti-CD3 antibody comprises the variable region of an antibody heavy chain. In some embodiments, an anti-CD3 antibody comprises one or more CDRs of an antibody light chain. In some embodiments, an anti-CD3 antibody comprises one or more CDRs of the antibody heavy chain of an anti-CD3 antibody. In some embodiments, an anti-CD3 antibody comprises one or more light chain CDRs and one or more heavy chain CDRs. In some embodiments, the anti-CD3 antibody comprises a Fab. In some embodiments, an anti-CD3 antibody comprises more than one Fab. In some embodiments, an anti-CD3 antibody comprises a scFv. In some embodiments, an anti-CD3 antibody comprises more than one scFv. In some embodiments, an anti-CD3 antibody comprises a minibody. In some embodiments, an anti-CD3 antibody comprises two or more minibodies. In some embodiments, an anti-CD3 antibody comprises a diabody. In some embodiments, an anti-CD3 antibody comprises more than one diabody. In some embodiments, the anti-CD3 antibody comprises a light chain variable region and a heavy chain variable region. In some embodiments, the anti-CD3 antibody comprises a complete light chain and a complete heavy chain. In some embodiments, an anti-CD3 antibody comprises one or more Fc domains or portions thereof. In some embodiments, the anti-CD3 antibody comprises any combination of the above embodiments. In some embodiments, the anti-CD3 antibody comprises a homodimer, heterodimer, homomultimer or heteromultimer of any of the above embodiments. In some embodiments, an anti-CD3 antibody comprises a polypeptide bound to a binding partner, wherein the binding partner is an antigen, polypeptide, nucleic acid molecule, polymer, or other molecule or substance. ). In some embodiments, an anti-CD3 antibody is conjugated to a scaffold molecule or substance other than the antibody.

In some embodiments, an anti-CD3 antibody has one or more post-translational modifications. In some embodiments, an anti-CD3 antibody is linked to a linker, polymer, or molecule with biological activity. In some embodiments, an anti-CD3 antibody is linked to a bifunctional polymer, bifunctional linker, or one or more additional anti-CD3 antibodies. In some embodiments, an anti-CD3 antibody is linked to a polypeptide that is not an anti-CD3 antibody. In some embodiments, the antigen-binding polypeptide having a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides (additional antigen-binding polypeptide may also have non-naturally encoded amino acids). In some embodiments, an antigen-binding polypeptide having a non-naturally encoded amino acid is linked to one or more polypeptide-small molecule conjugates (polypeptide-small molecule conjugates may also have non-naturally encoded amino acids). In some embodiments, the anti-CD3 antibody having a non-naturally encoded amino acid is linked to one or more additional antigen-binding polypeptides (additional antigen-binding polypeptides are also , may have non-naturally encoded amino acids).

In some embodiments, the non-naturally encoded amino acid is linked to a water soluble polymer. In some embodiments, the water-soluble polymer contains polyethylene glycol moieties. In some embodiments, polyethylene glycol molecules are bifunctional polymers. In some embodiments, a bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is an antigen binding polypeptide. In some embodiments, the second polypeptide is an anti-CD3 antibody.

In some embodiments, amino acid substitutions in the anti-CD3 antibody may be with naturally occurring amino acids or with non-naturally occurring amino acids. However, one or more of the substitutions are with non-naturally encoded amino acids.

In some embodiments, the non-naturally encoded amino acid has a carbonyl, acetyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide or alkyne group.

In some embodiments, the polyethylene glycol molecule has a molecular weight of about 0.1 kDa to about 100 kDa. In some embodiments, the polyethylene glycol molecule has a molecular weight between 0.1 kDa and 50 kDa.

In some embodiments, polyethylene glycol molecules are branched polymers. In some embodiments, the molecular weight of each branch of the polyethylene glycol branched polymer is 1 kDa to 100 kDa, or 1 kDa to 50 kDa.

The invention also provides an anti-CD3 antibody polypeptide comprising a linker, polymer or biologically active molecule attached to one or more non-naturally encoded amino acids (such as The missing amino acid is incorporated by the ribosome into the polypeptide at a preselected position).

We show that T cell attack on target cells and injury by anti-CD3 Fab-folate is dose dependent. FIG. 1(A) shows KB cells (FR ⁺ ), OV-90 cells (FR ⁺ ) and CAKI-1 cells (FR ⁻ ) upon treatment with anti-CD3Fab-folate in the presence of activated human PBMC. is dose dependent (target cells: effector cells = 1:10). Target cells were incubated for 12-24 hours at 37° C., 5% CO ₂ under increasing concentrations of anti-CD3 Fab-folate or unconjugated anti-CD3 Fab. Cytotoxicity was quantified by measuring LDH levels released from lysed cells according to the manufacturer's protocol (Cytotox 96 nonradioactive cytotoxicity assay, Promega). FIG. 1(B) shows SKOV-3 cells maintained in RPMI-1640 medium without folate. This medium was supplemented with 10% FBS for at least three passages before the cells were seeded into 48-well cell culture plates and allowed to adhere to the plates. Also, human PBMCs were added after cell attachment (target cells: effector cells=1:10). Anti-CD3 Fab or anti-CD3 Fab-folate was diluted in folate-free medium and added at the indicated concentrations and the co-medium was incubated for 16 hours. Images were taken with a 25x objective microscope. The in vivo efficacy of anti-CD3 Fab-folate bispecific agents was evaluated in a xenograft model using female NOD-SCID mice. Animals were fed a low-folate diet to reduce circulating serum folate levels that could compete with the bispecific drug. KB cells (FR ⁺ ) or A549 cells (FR ⁻ ) were mixed with non-activated human PBMCs (target cells:effector cells=1:100) or activated human PBMCs (target cells:effector cells=1:10). Tumorigenicity was observed when the mixture was injected subcutaneously into mice. Mice were implanted with KB cells (target cells:effector cells=1:10) mixed with human PBMCs. The mice were then injected intravenously with 1.5 mg/kg anti-CD3 Fab-folic acid or PBS (starting on the same day as tumor cell implantation and continued daily for 10 days). Figure 2(A) shows the tumor volume in this experiment. A rapid increase in tumor volume was seen in mice treated with PBS (approximately 5-day doubling rate). In contrast, tumors in mice treated with anti-CD3 Fab-folate were barely detectable throughout the study. In a parallel study, mice were implanted with KB cells mixed with non-activated human PBMC (target cells:effector cells=1:100). They were then treated intravenously with 1.5 mg/kg anti-CD3 Fab-folic acid or PBS (starting on the same day as tumor cell implantation and continued daily for 10 days). FIG. 2(B) shows the tumor volume in the experiment with target cells:effector cells=1:100. The rate of tumor reduction was faster in mice treated with anti-CD3 Fab-folate. From study day 35 onwards, tumors in PBS-treated mice gradually increased in volume. In contrast, tumors in mice treated with anti-CD3 Fab-folate decreased to a marginally detectable extent. This indicates that anti-CD3 Fab-folate conjugates eliminate tumors by attacking cytotoxic T cells even in the absence of activated T cells. FIG. 2(C) graphically depicts the change in body weight for mice in both treatment groups. FIG. 2(D) shows serum concentrations over time in the three test groups of rats. The test groups were a group injected with 1 mg/kg anti-CD3 Fab-folic acid; a group injected with 5 mg/kg anti-CD3 Fab-folic acid; and a group injected with 1 mg/kg anti-CD3 Fab. Serum was collected at regular intervals and analyzed by ELISA. Both anti-CD3 Fab-folate and the corresponding unconjugated anti-CD3 Fab variant decreased in serum concentrations at the same rate (serum half-life: 60 minutes). Binding of folate conjugates to FR and CD3. FIG. 3(A) is a graphical representation of the binding of folate-CF488 to KB cells (FR ⁺ ) but not to A549 cells (FR ⁻ ) (free folate (analyzed by competition with Cells were incubated for 30 minutes at 4° C. with medium containing 100 nM folate under an increasing series of complex concentrations. Cell-associated fluorescence was then analyzed by flow cytometry. FIG. 3(B) is a graphical representation of the binding of anti-CD3 Fab-CF488 to Jurkat cells (CD3 ⁺ ) but not to KB cells (CD3 ⁻ ). Cells were incubated for 30 minutes at 4° C. under an increasing series of complex concentrations. Binding was analyzed by flow cytometry. T-cell mediated injury of anti-CD3 Fab-folate conjugates by two independent cytotoxicity assays. Treatment with anti-CD3 Fab-folic acid in the presence of non-activated human PBMC (target cells: effector cells = 1:10) showed a dose-dependent T cell-mediated cytotoxicity in KB cells (FR ⁺ ). was observed in A549 cells (FR ⁻ ), whereas it was absent in A549 cells (FR − ). Incubated with target cells for 12-24 hours at 37° C., 5% CO ₂ under increasing concentrations of anti-CD3 Fab-folate. In FIG. 4(A), cells were labeled with DIO (staining the cell membrane green) and propidium iodide was added to a final concentration of 1 μg/mL. Cytotoxicity was measured by flow cytometry. FIG. 4(B) is a graphical representation of the ATP content of KB cells 24 hours after PBMC washout (measured using Cell Titer Glo (Promega)). Binding of humanized SP34 Fab and novel sequences (SEQ ID NOs: 1-15) included in the complex to SKOV-3. Assays were performed in media without folic acid. Binding of humanized SP34 Fab and novel sequences (SEQ ID NOs: 1-15) included in the complex to SKOV-3. Assays were performed in media containing 50 nM SFA. (a) shows CD69 expression induced by administration of chimerized SP34-folic acid. (b) shows survival of HPB-ALL cells treated with the same serum samples as in FIG. 7(a). Killing assay showing percent cytotoxicity in human PBMC. The conditions of 50 nM SFA in FIG. 6 were reproduced.

[Definition]
It is to be understood that this invention is not limited to the particular methods, protocols, cell lines, constructs and agents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. The scope of the invention is limited only by the appended claims.

As used in this specification and the appended claims, the singular forms ("a," "an," "the") include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide-small molecule complex" or "anti-CD3 antibody" is a reference to one or more of such proteins, including equivalents known to those skilled in the art. ing.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. However, preferred methods, devices and materials are those described herein.

Publications and patents mentioned in this specification are hereby incorporated by reference. This is for the purpose of description and disclosure (eg, of structures and methods described in that publication that may be used in connection with the present invention). The publications discussed herein merely provide their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate the above disclosure by virtue of prior invention or otherwise.

The term "substantially purified" means that the anti-CD3 antibody is normally associated with proteins in the environment in which it is naturally produced (i.e., natural cells) or host cells (in the case of recombinantly produced anti-CD3 antibodies). or substantially or essentially free of components that interact with proteins. Anti-CD3 antibodies substantially free of cellular material include, for example, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 15%, by dry weight of contaminating protein, about Included are preparations of protein containing less than 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%. When the anti-CD3 antibody or variant thereof is recombinantly produced from a host cell, such protein accounts for no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15% of the dry weight of the cell, It may be present at about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less. When the anti-CD3 antibody or variant thereof is recombinantly produced from a host cell, the amount of such protein in the medium is about 5 g/L or less, about 4 g/L or less, about 3 g/L or less of the dry weight of the cells. , about 2 g/L or less, about 1 g/L or less, about 750 mg/L or less, about 500 mg/L or less, about 250 mg/L or less, about 100 mg/L or less, about 50 mg/L or less, about 10 mg/L or less, or It may be present up to about 1 mg/L. Accordingly, the purity of a "substantially purified" anti-CD3 antibody when provided by the methods of the present invention is about 30% or higher, about 35% or higher, about 40% or higher, about 45% or higher, about 50% or higher, % or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more. Specifically, the purity may be about 75% or higher, about 80% or higher, about 85% or higher. More specifically, it may be about 90% pure or more, about 95% pure or more, about 99% pure or more, or more. Here, purity is determined by suitable methods (SDS/PAGE analysis, RP-HPLC, SEC and capillary electrophoresis, etc.).

A "recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide. The method of inserting the exogenous polynucleotide is irrelevant (eg, direct uptake, transduction, f-mating, or methods known in the art for making recombinant host cells). An exogenous polynucleotide may be maintained as a non-integrating vector (eg, a plasmid) or may otherwise be integrated into the host genome.

Antibodies are proteins that exhibit binding specificity for a particular antigen. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond. The number of disulfide bonds between heavy chains varies with immunoglobulin isotype. Each heavy and each light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain at one end (V _L ) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain. Also, the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.

The term "variable" refers to the significant variation in sequence of certain portions of the variable domains among antibodies. This sequence difference is responsible for the binding specificity of each particular antibody for a particular antigen. However, the variability is not evenly distributed among the variable domains of antibodies. The variability is concentrated in three regions called complementarity determining regions (CDRs) that are present in both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). Both the heavy and light chain variable domains in nature contain four FR regions. Many of them adopt a β-sheet structure and are linked to 3 or 4 CDRs. The CDRs form loop structures and in some cases form part of a β-sheet structure. The CDRs of each chain are held together in close proximity by the FR regions. Together with the CDRs of other chains, they contribute to the formation of the antibody's antigen-binding site (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

The constant domains are not directly involved in the binding of an antibody to antigen, but exhibit various effector functions. Depending on the amino acid sequence of their heavy chain constant region, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins (IgA, IgD, IgE, IgG and IgM), some of which can be further divided into subclasses (isotypes) (e.g. IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2). The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Among the various human immunoglobulin classes, only human IgG1, human IgG2, human IgG3 and human IgM are known to activate complement.

Affinity maturation of antibodies in vivo proceeds by antigen selection of high affinity antibody variants generated primarily by somatic hypermutation. A "repertoire shift" also usually occurs. In repertoire shift, the dominant germline genes of secondary or tertiary responses have been found to be different from those of primary and secondary responses.

The process of affinity maturation of the immune system can be recapitulated by in vitro mutagenesis of antibody genes and isolation of affinity-enhancing mutants by affinity selection. Such variant antibodies can be displayed on the surface of filamentous bacteriophages or microorganisms such as yeast. Antibodies can then be selected according to their affinity for the antigen or according to their dissociation rate (off-rate) from the antigen (Hawkins et al. J. Mol. Biol. 226:889-896 ( 1992)). Affinity maturation of a human antibody that binds to the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) has been achieved by CDR walking mutagenesis (Barbas III et al. PNAS (USA) 91: 3809- 3813 (1994); Yang et al. J. Mol. Biol. 254:392-403 (1995)). There is also a case where anti-c-erbB-2 single-chain Fv fragment was affinity-matured by a similar technique (Schier et al. J. Mol. Biol. 263: 551567 (1996)). There have been cases of affinity maturation of human antibodies with high affinity for the third hypervariable loop of HIV by antibody chain shuffling and CDR mutagenesis (Thompson et al. J. Mol. Biol. 256:77-88 (1996). )). [Balint and Larrick Gene 137:109-118 (1993)] discloses computational oligodeoxyribonucleotide-specific scanning mutagenesis. In this approach, the entire CDRs of variable region genes are searched simultaneously for improved variants. In some cases, an initial limited mutagenesis strategy was used to affinity mature a humanized antibody specific for αvβ3. In this approach, all six CDR positions are mutated, expressed, and screened to obtain a combinatorial library containing the mutants with the highest affinity (Wu et al. PNAS (USA) 95: 6037). -6-42 (1998)). Phage-displayed antibodies are reviewed in Chiswell and McCafferty TIBTECH 10:80-84 (1992); Rader and Barbas III Current Opinion in Biotech. 8:503-508 (1997). In each of the above-mentioned publications in which a variant antibody with improved affinity compared to the parent antibody was reported, the variant antibody had amino acid substitutions in the CDRs.

As used herein, "affinity maturation" refers to the process of improving the affinity of an antibody for an antigen. Methods of affinity maturation include, but are not limited to, computational screening methods and experimental methods.

By "antibody" herein is meant a protein comprising one or more of the polypeptides substantially encoded by all or part of antibody genes. The immunoglobulin genes include the constant region genes kappa, lambda, alpha, gamma (IgG1, IgG2, IgG3 and IgG4), delta, epsilon and mu, as well as the myriad immunoglobulin variable region genes, provided that these is not limited to). Antibodies herein include full-length antibodies and antibody fragments. Also included are antibodies naturally occurring in any organism, as well as artificially produced antibodies (eg, variants).

By "antibody fragment" is meant an antibody that is in any form other than the full-length form. Antibody fragments as used herein include (i) antibodies that are small portions contained in full-length antibodies, and (ii) artificially produced antibodies. Antibody fragments include but are not limited to Fv, Fc, Fab and (Fab') ₂ , single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, framework regions, constant regions, etc. (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9: 395-402).

As used herein, "computational screening method" means any method that designs one or more mutations in a protein. This method utilizes a computer to evaluate the interaction energy between possible amino acid side chain substitutions and/or the interaction energy between possible amino acid side chain substitutions and the rest of the protein.

By "Fc" is meant that portion of an antibody that is composed of the immunoglobulin Cγ2 and Cγ3 domains (Cγ2 and Cγ3). Fc may include any residue present in the N-terminal hinge between Cγ2 and Cγ1 (Cγ1). "Fc" may refer to the region in isolation or in an antibody or antibody fragment. Fc also includes any modified form of Fc. For example, native monomers, native dimers (disulfide-linked), modified dimers (disulfide-linked and/or non-covalently linked), and modified monomers (that is, derivatives), but are not limited to these.

As used herein, the term "full-length antibody" refers to the structures of the H and/or L chains of an antibody that make up its natural biological form. In many mammals (eg, humans and mice), this form is a tetramer, consisting of two pairs of two immunoglobulin chains (two identical pairs). Each combination contains one light chain and one heavy chain. Each light chain contains _the VL and _CL domains of an immunoglobulin. Each heavy chain contains the _VH , Cγ1, Cγ2 and Cγ3 domains of an immunoglobulin. Within each combination, the light and heavy chain variable regions (V _L and V _H ) are jointly responsible for binding to antigen. The constant regions (CL, Cγ1, Cγ2 and Cγ3, especially Cγ2 and Cγ3) are responsible for the effector functions of the antibody. In certain mammals (eg, camels and llamas), full-length antibodies may consist of only two heavy chains. Each heavy chain contains the _VH , Cγ2 and Cγ3 domains of an immunoglobulin.

As used herein, "immunoglobulin (Ig)" refers to a protein composed of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies and the like. Immunoglobulins may take a variety of structural forms. Examples include, but are not limited to, full-length antibodies, antibody fragments, individual domains of immunoglobulins (eg, but not limited to V _H , Cγ1, Cγ2, Cγ3, V _L and C _L ).

By "immunoglobulin (Ig) domain" herein is meant a protein domain consisting of a polypeptide substantially encoded by an immunoglobulin gene. Domains of Ig include, but are not limited to, _{V H} , Cγ1, Cγ2, Cγ3, VL and _CL (as shown in FIG. 1).

As used herein, "mutant protein sequence" means a protein sequence that has one or more residues that differ in amino acid identity from other similar protein sequences. The "similar protein sequence" mentioned above may be the naturally occurring wild-type protein sequence, or it may be a variant that differs from the wild-type sequence. We generally refer to the original array as the "parent array". A parental sequence may be a wild-type or mutant sequence. For example, in preferred embodiments of the invention, a humanized parental sequence may be utilized to generate variants by computational analysis thereof.

As used herein, the "variable region" of an antibody is composed of an immunoglobulin _VH domain, an immunoglobulin _VL domain, or an immunoglobulin _VH and _VL domain, as shown in FIG. , means one or more polypeptides (including variants). "Variable region" may refer to these polypeptides in their isolated state (as those regions contained in Fv fragments, scFv fragments, larger antibody fragments), full-length antibodies (or non-antibody It may refer to the relevant region contained in the scaffold molecule).

The present invention is applicable to antibodies obtained from a wide variety of sources. Antibodies can be substantially encoded by one or more antibody genes from any organism. Such organisms include humans, mice, rats, rabbits, camels, llamas, dromedaries, monkeys, particularly mammals, particularly humans, particularly mice and rats. (but not limited to). In one embodiment, the antibody is a fully human antibody. Fully human antibodies can be obtained, for example, from a patient or subject using transgenic mice or other animals (Bruggemann & Taussig, 1997, Curr. Opin. Biotechnol. 8:455-458), or human antibody libraries. and a selection method (Griffiths & Duncan, 1998, Curr. Opin. Biotechnol. 9:102-108). Antibodies may be derived from any source, including man-made and naturally occurring sources. For example, the invention may utilize artificial antibodies. Engineered antibodies include, but are not limited to, camelized and humanized antibodies (Clark, 2000, Immunol. Today 21:397-402)), or antibodies derived from combinatorial libraries. Furthermore, artificial variants of antibodies substantially encoded by one or more of the naturally occurring antibody genes may be optimized antibodies. For example, in one embodiment the optimized antibody is an antibody identified by affinity maturation.

With respect to the anti-CD3 antibodies of the invention, the term "antigen-specific" or "specifically binds" refers to one or more epitopes of a given antigen or to a given binding target in a sample containing a mixture containing the antigen. , but does not substantially recognize or bind other molecules.

As used herein, the term "bispecific anti-CD3 antibody" or "multispecific anti-CD3 antibody" refers to an anti-CD3 antibody having two or more antigen binding sites or binding partner binding sites. show. wherein the first binding site has an affinity for a first antigen or epitope and the second binding site has a binding affinity for a second antigen or epitope (different from the first antigen or epitope) have a sexuality.

As used herein, the term "epitope" refers to the site on an antigen or binding partner that is recognized by an anti-CD3 antibody. When the antigen comprises a polypeptide, the epitope can be a linear or sterically formed sequence or shape of amino acids. An epitope can also be any position on any type of antigen where an anti-CD3 antibody binds to that antigen.

As used herein, "antigen-binding polypeptides" or "anti-CD3 antibodies" refer to polypeptides and proteins that have at least the biological activity of specific binding to a specific binding target (such as an antigen). must be included. Also anti-CD3 antibody analogs, isoforms, anti-CD3 antibody mimetics, anti-CD3 antibody fragments, hybrid anti-CD3 antibody proteins, fusion proteins, oligomers and multimers of the above polypeptides and proteins. , homologues, glycosylation variants, and muteins, whether or not they possess similar biological activity, should also be included. Furthermore, the method of synthesizing or making these substances is also not limited. Such methods of synthesis or production include recombinant (whether produced from cDNA, genomic DNA, synthetic DNA or other forms of nucleic acid), by microinjection of nucleic acid molecules in vitro or in vivo. Methods include, but are not limited to, synthetic methods, transgenic methods, and gene activated methods. Specific examples of anti-CD3 antibodies include antibody molecules, heavy chains, light chains, variable regions, CDRs, Fabs, scFvs, other backbone non-antibody molecules, ligands, receptors, peptides, or any antibody that binds to an antigen. Amino acid sequences include, but are not limited to.

The term "anti-CD3 antibody" or "antigen-binding polypeptide" refers to a polypeptide that retains one or more of the biological activities of a native antibody in addition to the anti-CD3 antibody described above. Such biological activities include (but are not limited to) activities other than binding to an antigen. Activities other than binding to antigen include, but are not limited to, any activity or activities associated with Fc.

Antigen-binding polypeptides include pharmaceutically acceptable salts and prodrugs. Prodrugs include salts, polymorphs, hydrates, solvates, biologically active fragments, biologically active variants, and stereoisomers of native human anti-CD3 antibodies. Also salts, polymorphs, etc. to agonist, mimetic and antagonist variants of naturally occurring human anti-CD3 antibodies, and polypeptide fusions thereof, are such prodrugs. Fusions with additional amino acids at the amino terminus, carboxy terminus, or both are also included in the term "antigen-binding polypeptide." Exemplary fusions include, but are not limited to, methionyl anti-CD3 antibodies, where a methionine is linked to the N-terminus of the anti-CD3 antibody as a result of recombinant expression. Also, fusions for purification purposes (e.g., but not limited to polyhistidine or affinity epitopes), fusions for linking anti-CD3 antibodies to other biologically active molecules, serum albumin binding Also included, but not limited to, are fusions with peptides, fusions with serum proteins (such as serum albumin).

The term "antigen" or "binding partner" refers to the substance to which the binding activity of an anti-CD3 antibody is targeted. Virtually any substance can be the antigen or binding partner of an anti-CD3 antibody.

Bispecific antibodies of the invention include anti-CD3 bispecific antibodies that are humanized SP34 antibodies or antibody fragments. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 160 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 172 of the light chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 157 of the light chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 129 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 129 of the heavy chain and position 172 of the light chain.

In some embodiments, the invention includes humanized SP34 antibodies that have a non-naturally encoded amino acid at position 204 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 210 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at 191 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 187 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 133 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 114 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 115 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 111 of the light chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 115 of the light chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 204 of the light chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 191 of the light chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 193 of the light chain. In some embodiments of the invention, a humanized SP34 antibody has a non-naturally encoded amino acid at position 186 of the light chain. In some embodiments of the invention, the humanized SP34 antibody has two of the above amino acid mutations. In some embodiments of the invention, the humanized SP34 antibody has two or more of the above amino acid mutations.

Bispecific antibodies of the invention include anti-CD3 bispecific antibodies that are humanized SP34 antibodies or antibody fragments. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Thr at position 160 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Lys at position 172 of the light chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Leu at position 157 of the light chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Lys at position 129 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has Lys at position 129 of the heavy chain and Lys at position 172 of the light chain replaced with a non-naturally encoded amino acid. In some embodiments of the invention, the humanized SP34 antibody has Lys at position 129 in the heavy chain and Leu at position 157 in the light chain replaced with a non-naturally encoded amino acid.

In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Asn at position 204 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Lys at position 210 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Thr at position 191 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Ser at position 187 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Gly at position 133 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Ala at position 114 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Ser at position 115 of the heavy chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Arg at position 111 of the light chain. In some embodiments of the invention, the humanized SP34 antibody has Ala at position 115 of the light chain replaced with a non-naturally encoded amino acid. In some embodiments of the invention, the humanized SP34 antibody has Leu at position 204 of the light chain replaced with a non-naturally encoded amino acid. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Lys at position 191 of the light chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Lys at position 193 of the light chain. In some embodiments of the invention, the humanized SP34 antibody has a non-naturally encoded amino acid substituted for Lys at position 186 of the light chain.

Bispecific antibodies of the invention include anti-CD3Fab-folate bispecific antibodies that are humanized SP34 antibodies or antibody fragments conjugated to folate. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Thr at position 160 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Lys at position 172 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Leu at position 157 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Lys at position 129 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Lys at position 129 of the heavy chain and Lys at position 172 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Lys at position 129 of the heavy chain and Leu at position 157 of the light chain.

In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Asn position 204 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Lys at position 210 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Thr at position 191 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Ser-187 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Gly at position 133 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Ala at position 114 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Ser at position 115 of the heavy chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Arg position 111 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Ala at position 115 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Leu at position 204 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folate at Lys at position 191 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Lys at position 193 of the light chain. In some embodiments of the invention, a humanized SP34 antibody is conjugated with folic acid at Lys at position 186 of the light chain.

Various publications disclose modification of polypeptides by conjugation with polymers or glycosylation. The term "anti-CD3 antibody" or "antigen-binding polypeptide" includes a polypeptide conjugated to a polymer such as PEG that contains cysteine, lysine, an N-terminal or C-terminal amino acid, or other residue Polypeptides, one or more of which may be additionally derivatized, include (but are not limited to). In addition, the anti-CD3 antibody may have a linker, polymer or biologically active molecule. Here, the amino acid conjugated with the linker, polymer or molecule having biological activity may be an unnatural amino acid according to the present invention. Alternatively, they may be conjugated to naturally-encoded amino acids using known techniques (such as coupling with lysine or cysteine). US Pat. No. 4,904,584 discloses PEGylated polypeptides with reduced lysines. The PEGylated polypeptide has one or more lysine residues deleted or substituted with other amino acid residues. WO 99/67291 discloses methods for conjugating proteins with PEG. In the above methods, one or more amino acid residues of the protein are deleted and the protein is contacted with PEG under conditions sufficient for protein conjugation. WO99/03887 discloses pegylated variants of polypeptides belonging to the growth hormone superfamily. Cysteine residues in the PEGylated variants are replaced with non-essential amino acid residues located in specific regions of the polypeptide. WO 00/26354 discloses methods for producing glycosylated polypeptide variants with reduced allergenicity. The polypeptide variants described above have one or more additional glycosylation sites compared to the corresponding parent polypeptide.

The term "antigen-binding polypeptide" also includes glycosylated anti-CD3 antibodies. Glycosylated anti-CD3 antibodies are polypeptides that are glycosylated at any amino acid position, such as N-linked glycosylated or O-linked glycosylated polypeptides (but not limited to not). Variants with a single nucleotide change are also considered biologically active anti-CD3 antibody variants. Furthermore, splice variants are also included in antigen-binding polypeptides. The term "antigen-binding polypeptide" also includes heterodimers, homodimers, heteromultimers or homomultimers of any one or more anti-CD3 antibodies. Alternatively, the antigen-binding polypeptide may be linked by chemical means to any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or any other type of biologically active molecule. Also included are substances expressed as fusion proteins. Also included are polypeptide analogs, eg, with specific deletions or other modifications, which still retain biological activity.

In some embodiments, the antigen-binding polypeptide further has additions, substitutions or deletions that modulate the biological activity of the anti-CD3 antibody. For example, additions, substitutions or deletions may modulate one or more properties or activities of the anti-CD3 antibody. Examples of such modulation include (i) modulation of affinity for antigen, (ii) modulation of antigen conformation or other secondary, tertiary, or quaternary structural changes (e.g., by increasing or decreasing the amount of change). (iii) stabilization of antigen conformation or other secondary, tertiary or quaternary structural changes; (iv) antigen conformation or other secondary structure; (v) regulation of circulating half-life; (vi) regulation of therapeutic half-life; (vii) regulation of polypeptide stability; (viii) regulation of dosage; (ix) modulating release or bioavailability; (x) enhancing purification; (xi) improving or altering a particular route of administration; Similarly, antigen-binding polypeptides may include protease cleavage sequences, reactive groups, antibody binding domains (eg, but not limited to FLAG or poly-His), or other affinity sequences (eg, FLAG, poly-His). His, GST, etc., but not limited to), or a binding molecule (eg, but not limited to biotin). Such binding molecules may facilitate detection of the polypeptide (eg, but are not limited to GFP), facilitate purification, or improve other attributes.

The term "antigen-binding polypeptide" also includes linked anti-CD3 antibody homodimers, heterodimers, homomultimers and heteromultimers. Modes of linkage include (i) direct linkage via side chains of non-naturally encoded amino acids, (ii) linkages between side chains of the same or different non-naturally encoded amino acids, and (iii) (iv) linked by fusion or (v) linked indirectly via a linker, including but not limited to linkage between the side chains of naturally encoded amino acids. Representative examples of linkers include, but are not limited to, small organic compounds, water-soluble polymers of various lengths, such as polyethylene glycols of various lengths, polydextrans or polypeptides.

Those skilled in the art understand that amino acid positions corresponding to positions in a specific antigen-binding polypeptide sequence can be easily identified in fragments of the antigen-binding polypeptide or related antigen-binding polypeptides. would do. For example, a sequence alignment program (such as BLAST) can be used to align and identify specific positions in a protein that correspond to positions in related sequences.

The term "antigen-binding polypeptide" also includes antigen-binding polypeptides having one or more amino acid substitutions, additions or deletions. Antigen-binding polypeptides of the invention may have modifications in which one or more natural amino acids are conjugated to one or more non-natural amino acids. Representative examples of substitutions at various amino acid positions in native anti-CD3 antibody polypeptides have already been described. Examples include (but are not limited to) substitutions that modulate one or more of the biological activities of the antigen-binding polypeptide. Specific examples include (but are not limited to) enhancement of agonist activity, enhancement of polypeptide solubility, conversion of polypeptides to antagonists, and the like. These examples are also included in the term "anti-CD3 antibody".

"Non-naturally encoded amino acid" refers to an amino acid that is not one of the twenty common amino acids or pyrrolysine or selenocysteine. Terms that can be used synonymously with the term "non-naturally encoded amino acid" include "non-natural amino acid," "unnatural amino acid," "non-naturally- occurring amino acids")", and hyphenated and unhyphenated forms of the above words. The term "non-naturally encoded amino acid" includes modifications (e.g., post-translational modifications) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine). It also includes (but is not limited to) amino acids that are produced by , but are themselves not normally incorporated into a growing polypeptide chain by a translation complex. Examples of such unnatural amino acids include (but are not limited to) N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine and O-phosphotyrosine.

"Amino terminal modification group" refers to any molecule that can be added to the amino terminus of a polypeptide. Similarly, a "carboxy terminal modification group" refers to any molecule that can be added to the carboxy terminus of a polypeptide. Terminal modification groups include, but are not limited to, various water-soluble polymers, peptides or proteins (such as serum albumin), or other moieties that extend the serum half-life of peptides.

The terms "functional group", "active site", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive site" are used in the art and herein to distinguish between Used to denote possible and definable parts or units. These terms have roughly the same meaning in the chemical arts. Also used herein to indicate a portion of a molecule that has some function or activity or that reacts with another molecule.

As used herein, the term "linkage" or "linker" is used to denote a group or bond, usually formed as a result of a chemical reaction and usually a covalent bond. By "hydrolytically stable linkage" is meant that the linkage is substantially stable in water and does not react with water at practical pH values (e.g., but not limited to, physiological unresponsive (possibly indefinitely) under adverse conditions). "Hydrolytically labile bond" or "hydrolytically degradable bond" means that the bond is degradable in water or an aqueous solution (eg, in blood). "Enzyme-labile bond" or "enzyme-labile bond" means that the bond can be cleaved by one or more enzymes. As is known in the art, PEG and related polymers have linker groups (i) in the polymer backbone or (ii) between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. It may have a degradable bond in it. For example, an ester bond formed by reaction of a PEG carboxylic acid or an activated PEG carboxylic acid with an alcohol group of a bioactive agent typically hydrolyzes under physiological conditions to release the agent. Other hydrolytically degradable bonds include (i) a carbonate bond, (ii) an imine bond resulting from the reaction of an amine with an aldehyde, and (iii) a phosphorus bond formed by the reaction of an alcohol with a phosphate group. acid ester bond, (iv) hydrazone bond, reaction product of hydrazide and aldehyde, (v) acetal bond, reaction product of aldehyde and alcohol, (vi) orthoester bond, reaction product of formate and alcohol (vii) an amine group (e.g., but not limited to, at the end of a polymer such as PEG) and a peptide bond formed by a carboxy group of a peptide; (viii) a phosphoramidite group (e.g., at the end of a polymer); but are not limited to) and oligonucleotide linkages formed by the 5' hydroxyl group of the oligonucleotide. Branched linkers may be used in the antigen-binding polypeptides of the present invention.

As used herein, the terms “biologically active molecule,” “biologically active portion,” or “biologically active agent” refer to biological systems, biological pathways, biomolecules, or organisms (e.g., viruses, bacteria, , bacteriophages, transposons, prions, insects, fungi, plants, animals and humans). It means any substance that can be given. Specifically, as used herein, "biologically active molecule" includes (i) any substance intended for the diagnosis, therapy, mitigation, treatment or prevention of disease in humans or other animals; or (ii) any substance intended to improve the physical or mental welfare of humans or animals, including but not limited to. Examples of biologically active molecules include peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, dyes, lipids, nucleosides, oligonucleotides, toxins, cells, viruses, liposomes. , microparticles and micelles. Classes of biologically active agents suitable for use in the present invention include drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiac Vasoactive agents, anti-anxiety agents, hormones, growth factors, steroidal agents, microbial-derived toxins, etc., include (but are not limited to).

The anti-CD3 antibodies of the invention may be conjugated with molecules such as PEG to improve their delivery and pharmacokinetic profiles in vivo. Leong et al. showed that site-specific PEGylation of a Fab′ fragment of an anti-IL-8 antibody reduced the clearance rate over that of the non-PEGylated state with little (or no) loss of antigen-binding activity. (Leong, S.R. et al. (2001) Cytokine 16:106-119).

Many other cleavable linkers are known to those of skill in the art (see US Pat. Nos. 4,618,492, 4,542,225 and 4,625,014). Mechanisms by which drugs are released from these linker groups include, for example, photoirradiation of photolabile bonds and acid-catalyzed hydrolysis. For example, US Pat. No. 4,671,958 describes immunoconjugates having linkers that are cleaved at target sites in vivo by proteases of the patient's complement system. The length of the linker may be predetermined or may be selected according to the desired spatial relationship between the anti-CD3 antibody and the molecule linked thereto. Numerous reports have been published on methods of attaching various radiodiagnostic, radiotherapeutic compounds, drugs, toxins and other agents to antibodies, and in view of this, one of ordinary skill in the art would be able to identify a given agent. Appropriate methods of attachment to anti-CD3 antibodies or other polypeptides can be determined.

"Bifunctional polymer" refers to a polymer having two separate functional groups. This functional group can specifically react with other moieties, including but not limited to side chains of amino acids, to form covalent or non-covalent bonds. (i) one functional group capable of reacting with a group possessed by a specific biologically active component; and (ii) the other group capable of reacting with a group possessed by a second biological component. A functional linker may be used to form a conjugate comprising a first biologically active moiety, a bifunctional linker and a second biologically active moiety. Many techniques and linker molecules are known for attaching various compounds to peptides. For example, European Patent Application No. 188,256, U.S. Pat. See 4,680,338, 4,569,789 and 4,589,071 (incorporated herein by reference). "Multifunctional polymer" refers to a polymer having two or more separate functional groups. This functional group can specifically react with other moieties, including but not limited to side chains of amino acids, to form covalent or non-covalent bonds. The bifunctional or multifunctional polymer can be of any desired molecular length or weight. Also, one may choose to provide a desired and specific spacing or configuration between the molecules linked to the anti-CD3 antibody.

As used herein, the term "water-soluble polymer" refers to any polymer that dissolves in an aqueous solvent. Conjugation of a water soluble polymer with an anti-CD3 antibody results in changes such as, but not limited to: (i) prolongation or modulation of serum half-life when compared to unmodified counterparts; , or to extend or modulate therapeutic half-life, (ii) modulate immunogenicity, (iii) modulate physical association properties (such as aggregation and multimerization), (iv) alter binding to receptors. , and modification of receptor dimerization or multimerization. A water-soluble polymer may or may not have bioactivity per se. A water-soluble polymer may also be used as a linker for adding other substances to the anti-CD3 antibody. Other substances include, but are not limited to, one or more anti-CD3 antibodies, or one or more biologically active molecules. Suitable polymers include, but are not limited to: polyethylene glycol, polyethylene glycol-propionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives of polyethylene glycol (US Pat. No. 5,252,714). incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether-maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, Dextran derivatives (such as dextran sulfate), polypropylene glycol, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (e.g. methylcellulose) and carboxymethyl cellulose), starch and starch derivatives, polypeptides, polyalkylene glycol and its derivatives, polyalkylene glycol copolymers and its derivatives, polyvinyl ethyl ether, α-β-poly[( 2-hydroxyethyl)-DL-aspartamide, etc., or mixtures thereof. Examples of such water-soluble polymers include (but are not limited to) polyethylene glycol and serum albumin.

As used herein, the term "polyalkylene glycol," "poly(alkene glycol)" refers to polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. The term "polyalkylene glycol" includes both linear and branched polymers. The average molecular weight of polyalkylene glycol is 0.1 kDa to 100 kDa. For example, commercial manufacturers' catalogs (such as Shearwater Corporation's catalog "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001)) summarize other exemplary embodiments.

As used herein, the term "modulated serum half-life/modulates serum half-life" means positive or negative in the circulating half-life of a modified anti-CD3 antibody compared to an unmodified one. means a change in Serum half-life is measured by taking blood samples at various time points after anti-CD3 antibody administration and determining the concentration of the molecule in question in each sample. Since serum concentration and time are correlated, serum half-life can be calculated. Although serum half-life extensions of about 2-fold or more are desirable, smaller serum half-life extensions may also be useful, for example, to provide adequate dosing regimens or to prevent toxic effects. In some embodiments, the serum half-life extension is about 3-fold or more, about 5-fold or more, or about 10-fold or more.

As used herein, the term "modulated therapeutic half-life/modulating therapeutic half-life" refers to a therapeutically effective amount of (i) an anti-CD3 antibody, or (ii) A positive or negative change in half-life of an anti-CD3 antibody bearing a modified biologically active molecule compared to an unmodified one. Therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. It is desirable to extend the therapeutic half-life to confer certain advantages on the dosing regimen or total dosage, or to avoid unwanted effects. In some embodiments, the therapeutic effect is achieved by enhancing efficacy, enhancing or weakening the binding of the modified molecule to its target, or increasing or decreasing other parameters or mechanisms of action of the unmodified molecule. half-life is prolonged.

The term "isolated/isolated" when applied to a nucleic acid or protein means substantially free of other cellular components with which the nucleic acid or protein is naturally associated. This can be a homogeneous state. The isolated material may be in a dry state, a semi-dry state, or a solution (including, but not limited to, an aqueous solution). Techniques of analytical science (such as polyacrylamide gel electrophoresis or high performance liquid chromatography) are typically used to determine purity and homogeneity. A protein that predominates among the species present in a preparation is substantially purified. Specifically, an isolated gene is separated from open reading frames that flank the gene and encode proteins other than those of the gene. The term "purified/purified" means that the nucleic acid or protein appears substantially as one band in an electrophoresis gel. Specifically, "purified" means that the nucleic acid or protein is 85% or greater, 90% or greater, 95% or greater, 99% or greater, or greater.

The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides, and polymers thereof. A "nucleic acid" may be in single-stranded or double-stranded form. Unless specifically limited, the term also includes nucleic acids containing known analogues of natural nucleotides. This analog has binding properties comparable to those of the nucleic acids to which it is compared and is metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs such as PNAs (peptide nucleic acids), DNA analogs used in antisense technology such as thiophosphates, amidophosphates, and the like. Unless otherwise stated, a particular nucleic acid sequence may have (i) conservatively modified variants (e.g., substitutions with degenerate codons, but , but not limited to), and (ii) complementary sequences are also substantially included. Specifically, substitution with codons that are degenerate produces sequences in which the third position of one or more (or all) selected codons is substituted with mixed bases and/or deoxyinosine residues. (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. ( 1992); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. Thus, references to polypeptides apply equally to references to peptides and to proteins (and vice versa). The term applies to both naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the term includes amino acid chains of any length (eg, full-length proteins whose amino acid residues are linked by covalent peptide bonds (ie, antigens)).

The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. The naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine). , tryptophan, tyrosine and valine), and pyrrolysine and selenocysteine. "Amino acid analogue" refers to a compound with the same basic chemical structure as a naturally occurring amino acid (i.e., hydrogen, carboxyl group, amino group, and the alpha carbon bonded to the R group) (homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc.). Such analogs have modified R groups (such as norleucine) or have modified peptide backbones. However, the same basic chemical structure as naturally occurring amino acids is preserved.

In this specification, amino acids may be represented by the generally known three-letter code, or by the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be designated by their commonly used one-letter symbols.

"Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode identical or essentially identical amino acid sequences. Where the nucleic acid does not encode an amino acid sequence, it represents essentially the same sequence. Due to the degeneracy of the genetic code, there are many functionally identical nucleic acids encoding any given protein. For example, the codons GCA, GCC, GCG and GCU all code for the amino acid alanine. Thus, wherever a codon specifies alanine, that codon can be changed to any of the corresponding codons described above without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. By every nucleic acid sequence that encodes a polypeptide herein is meant all possible silent variations of that nucleic acid. Those skilled in the art will appreciate that each codon in a nucleic acid can be modified to yield a functionally identical molecule (usually AUG, the only codon for methionine, and usually the only codon for tryptophan). (except TGG, which is Accordingly, substantially each silent variation of a nucleic acid which encodes a polypeptide is included in each described sequence.

For amino acid sequences, when individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence alter, add or delete one or a small percentage of amino acids in the encoded sequence. are also "conservatively modified variants" (if the change results in the substitution of an amino acid with a chemically similar amino acid). Conservative substitution lists providing functionally similar amino acids are well known in the art. In addition, such conservatively modified variants also include polymorphic variants, interspecies homologs and alleles of the invention.

Each of the following eight groups describes amino acids that are conservatively substituted for each other:
(1) alanine (A), glycine (G);
(2) aspartic acid (D), glutamic acid (E);
(3) asparagine (N), glutamine (Q);
(4) arginine (R), lysine (K);
(5) isoleucine (I), leucine (L), methionine (M), valine (V);
(6) phenylalanine (F), tyrosine (Y), tryptophan (W);
(7) Serine (S), Threonine (T);
(8) cysteine (C), methionine (M)
(See, eg, Creighton, Proteins: Structures and Molecular Properties (WH Freeman &Co.; 2nd edition (December 1993))).

As used herein, the term "subject" refers to an animal subject to treatment, observation or experimentation, preferably a mammal, and most preferably a human.

As used herein, the term "effective amount/effective amount" means that one or more of the symptoms of the disease, condition or disorder to be treated is represents a reduction to some extent. Compositions containing the (modified) non-natural amino acid polypeptides described herein can be administered for prophylactic, enhancing and/or therapeutic treatments.

The term "enhance," "enhancing" means to increase or prolong, either in strength or duration, a desired effect. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhancing/enhancing" means increasing or prolonging, either in strength or duration, the effect of another therapeutic agent in a system. represent ability. As used herein, an "enhancing effective amount" refers to an amount sufficient to enhance the effect of a therapeutic agent in a desired system. When used in a patient, amounts effective for such use will depend on the severity and course of the disease, disorder or condition, prior treatment, the patient's state of health and response to drugs, and the judgment of the treating physician.

As used herein, the term "modified/modified" refers to the presence of post-translational modifications in a polypeptide. A form of the term "(modified)" means that the polypeptide under discussion has been modified in any configuration. Thus, the polypeptides discussed may be modified or unmodified.

The terms "post-translationally modified" and "modified" refer to any modification of a natural or non-natural amino acid that occurs after the amino acid is incorporated into a polypeptide chain. The term includes, by way of example only, co-translational modifications in vivo, post-translational modifications in vivo, and post-translational modifications in vitro.

In therapeutic applications, a composition containing the (modified) non-natural amino acid polypeptide is administered to a patient already afflicted with a disease, condition or disorder in a therapeutically sufficient amount (or at least only an amount that partially suppresses the symptoms of the disease, disorder or condition). Such an amount is defined as a "therapeutically effective amount." A therapeutically effective amount will depend on the severity and course of the disease, disorder or condition, prior treatment, the patient's state of health and response to drugs, and the judgment of the treating physician. It is understood in the art that such therapeutically effective amounts can be determined by routine experimentation (eg, a dose escalation clinical trial).

The term "treatment" is used to refer to both prophylactic and/or therapeutic treatment.

Unless otherwise stated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacy within the skill of the art are employed.

[Detailed description of the invention]
[Introduction]
The present invention provides anti-CD3 antibody molecules having one or more unnatural amino acids. In certain embodiments of the invention, the anti-CD3 antibody with one or more unnatural amino acids has one or more post-translational modifications. In one embodiment, the one or more post-translational modifications include molecular additions. The molecules to be attached at this time include water-soluble polymers, polyethylene glycol derivatives, drugs, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, biologically active agents, small molecules, or any of the above. Including, but not limited to, combinations, or any other desired compounds or substances. These molecules have a second reactive group and react with the one or more unnatural amino acids that have the first reactive group. This reaction is by chemical methods known to those skilled in the art to be suitable for the particular reactive group. For example, the first reactive group is an alkynyl moiety (such as, but not limited to, that in the unnatural amino acid p-propargyloxyphenylalanine (propargyl groups are sometimes also referred to as acetylene moieties)). , the second reactive group is the azide moiety. In this example, a [3+2] cycloaddition chemical method is utilized. In another example, the first reactive group is an azide moiety (such as, but not limited to, in the unnatural amino acid p-azido-L-phenylalanine) and the second reactive group is It is an alkynyl moiety. In certain embodiments of the modified anti-CD3 antibody polypeptides of the invention, one or more non-natural amino acids having one or more post-translational modifications at the sugar moieties with one or more post-translational modifications (eg, but not limited to, unnatural amino acids with keto functional groups) have been used. In certain embodiments, post-translational modifications are made in vivo in eukaryotic or non-eukaryotic cells.

Table 1 lists novel humanized variable heavy chain sequences, humanized variable light chain sequences, and humanized variable heavy chain sequences and humanized variable light chain sequences of anti-CD3 antibodies of the invention.

The antigen-binding polypeptide and small molecule may be linked by a linker, polymer, or covalent bond. The linker, polymer or small molecule may itself have functional groups that do not react with the 20 common amino acids. A linker or polymer may be bifunctional. One or more bonds involved in linking the antigen-binding polypeptide to the biologically active molecule via a linker, polymer or covalent bond may be irreversible or reversible under desired circumstances. may be unstable. The one or more bonds involved in linking the antigen-binding polypeptide to the molecule via a linker, polymer or covalent bond may modulate the release of the antigen-binding polypeptide or other molecule. good. Various small molecules may be produced by chemical means, isolated as natural products, or produced by other means by those skilled in the art.

[Rader et al. in Proc Natl Acad Sci US A. 2003 Apr 29;100(9):5396-400] confer effector functions on synthetic small molecules by reacting them with generic antibody molecules, Disclosed are methods for extending serum half-life (incorporated herein by reference). The conjugates disclosed in the above references link between mAb 38C2, a catalytic antibody that mimics the natural aldolase enzyme, and a diketone derivative of an integrin-targeted Arg-Gly-Asp peptidomimetic. Created by reversible covalent attachment through reactive lysine residues. Not only did the peptidomimetic have an extended half-life, but the conjugate also has a retargeting switch to selectively target cell surfaces expressing integrins α _{v β3} and α _{v β5} . Admitted.

The present invention provides methods and compositions based on antigen-binding polypeptides (or anti-CD3 antibodies) having one or more non-naturally encoded amino acids. The introduction of one or more non-naturally encoded amino acids into an anti-CD3 antibody has enabled the application of conjugation chemistry involving specific chemical reactions. Such chemical reactions can occur, for example, with one or more non-naturally encoded amino acids, but not with the 20 commonly occurring amino acids, including but not limited to not). In some embodiments, an anti-CD3 antibody having a non-naturally encoded amino acid is attached to a water-soluble polymer (such as polyethylene glycol (PEG)) via the side chain of the non-naturally encoded amino acid. is connected with The present invention provides a highly efficient method for selectively modifying proteins with PEG derivatives. This method involves the selective incorporation of non-gene-encoded amino acids. Non-gene-encoded amino acids include functional groups or substituents (e.g., ketone, azide, or acetylene moieties) not found in the 20 amino acids normally incorporated into proteins in response to selected codons. , but are not limited to. As a result, the amino acid is appropriately reacted with the PEG derivative to be modified. Once incorporated, the amino acid side chains are then modified, by suitable chemical methods known to those of skill in the art, to specific functional groups or substituents found in naturally encoded amino acids. can be done. A wide variety of known chemical methods are suitable for use in the present invention to incorporate water-soluble polymers into proteins. Such methods include, but are not limited to, Huisgen [3+2] cycloaddition reactions (e.g., [Padwa, A. in Comprehensive Organic Synthesis , Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p. 1-176]).

Since the Huisgen [3+2] cycloaddition method involves cycloaddition rather than nucleophilic substitution reactions, proteins can be modified with very high selectivity. This reaction can occur at room temperature under aqueous conditions with excellent regiospecificity (1,4>1,5) by adding a catalytic amount of a Cu(I) salt to the reaction mixture (e.g. Chem. 67:3057-3064; Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599] and WO03/ 101972). Molecules that can be added to proteins of the invention by [3+2] cycloaddition include, but are not limited to, azide or acetylene derivatives, substantially includes all molecules. These molecules are either unnatural amino acids with an acetylene group (such as, but not limited to, p-propargyloxyphenylalanine) or unnatural amino acids with an azide group (such as p-azido-phenylalanine, but (not limited to this) can be added respectively.

The five-membered ring resulting from the Huisgen [3+2] cycloaddition is generally irreversible in reducing environments and is hydrolytically stable for long periods of time, even in aqueous environments. Therefore, the physical and chemical properties of a wide variety of substances can be modified by the activated PEG derivatives of the invention, even in harsh aqueous environments. More importantly, because the azide and acetylene moieties are specific to each other (and, for example, do not react with any of the 20 common gene-encoded amino acids), one or more specific proteins can be modified at specific sites with very high selectivity.

The present invention also provides water-soluble and hydrolytically stable derivatives of PEG derivatives and related hydrophilic polymers having one or more acetylene or azide moieties. PEG polymer derivatives having acetylene moieties couple with high selectivity to azide moieties selectively introduced into proteins corresponding to selected codons. Similarly, PEG polymer derivatives bearing azide moieties couple with high selectivity to acetylene moieties selectively introduced into proteins corresponding to selected codons.

More specifically, azide moieties include, but are not limited to, alkylazides, arylazides, and derivatives of these azides. The alkylazide and arylazide derivatives may have other substituents as long as the acetylene-specific reactivity is preserved. Acetylene moieties include alkylacetylenes and arylacetylenes, and derivatives thereof. The alkylacetylene and arylacetylene derivatives may have other substituents as long as the azide-specific reactivity is preserved.

Accordingly, an anti-CD3 antibody is intended to encompass any polypeptide exhibiting specific binding ability to a target molecule or antigen. Any known antibody or antibody fragment is an anti-CD3 antibody.

The anti-CD3 antibodies of the invention may comprise an Fc region or Fc-like region. The Fc domain provides association with effector functions such as complement or phagocytic cells. The Fc portion of immunoglobulins has a long plasma half-life, whereas Fabs are short-lived (Capon, et al. (1989), Nature, 337:525-531). Fc domains can confer a longer half-life when assembled with therapeutic proteins. Alternatively, functions such as binding to Fc receptors, protein A binding, complement fixation, and possibly placental crossing can be provided. For example, the Fc region of an IgG1 antibody is N-terminal to CD30-L, a molecule that binds Hodgkin's disease tumor cells, anaplastic lymphoma cells, T-cell leukemia cells, and other malignant cell types expressing the CD30 receptor. (U.S. Pat. No. 5,480,981). IL-10, an anti-inflammatory and anti-rejection agent, has been fused to murine Fcγ2 to prolong the short circulating half-life of this cytokine (Zheng, X. et al. (1995), The Journal of Immunology, 154: 5590-5600). Studies have also shown that tumor necrosis factor receptor linked to human IgG1 Fc protein was used to treat septic shock patients (Fisher, C. et al., N. Engl. J. Med., 334: 1697- 1702 (1996); Van Zee, K. et al., The Journal of Immunology, 156: 2221-2230 (1996)) and rheumatoid arthritis patients (Moreland, et al. (1997), N. Engl. J. Med. , 337(3):141-147) has also been investigated. Fc has also been fused to the CD4 receptor to create therapeutic proteins for treating AIDS (Capon et al. (1989), Nature, 337:525-531). Furthermore, to ameliorate the short half-life and systemic toxicity of interleukin-2, the N-terminus of interleukin-2 has been fused to the Fc portion of IgG1 or IgG3 (Harvill et al. (1995), Immunotechnology 1:95-105).

It is well known that the Fc region of an antibody is composed of a monomeric polypeptide unit. The monomers can be linked into dimeric or multimeric forms by disulfide bonds or covalent associations. The number of intermolecular disulfide bonds that exist between the monomeric subunits of a native Fc molecule varies depending on the antibody class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). , There are 1 to 4 openings. As used herein, the term "Fc" is a generic term for monomeric, dimeric, and multimeric forms of the Fc molecule. Note that Fc monomers will spontaneously dimerize in the presence of suitable Cys residues, unless specific conditions prevent dimerization by formation of disulfide bonds. Even if the Cys residues in the Fc dimer that normally form disulfide bonds have been removed or replaced with other residues, the monomer chains are normally non-covalently interconnected. Dimerize by action. As used herein, the term "Fc" is used to mean all of the following forms: naturally occurring monomers, naturally occurring dimers (disulfide bond linked), modified dimers (disulfide bond and /or non-covalently linked), and modified monomers (ie derivatives).

For example, variants, analogs or derivatives of the Fc portion may be constructed by variously substituting residues or creating sequences with non-naturally encoded amino acids. Polypeptide variants (or analogs) include insertional variants, in which one or more amino acid residues are added to the Fc amino acid sequence. Insertions may be located at one or both ends of the protein, or may be located in intermediate regions of the Fc amino acid sequence. Insertional variants with additional residues at one or both ends can include, for example, fusion proteins and proteins with amino acid tags or labels. For example, an Fc molecule may optionally have a Met at the N-terminus, especially if the molecule is recombinantly expressed in bacterial cells such as E. coli. In deletion variants of Fc, one or more amino acid residues in the Fc polypeptide are removed. One or both termini of the Fc polypeptide may be deleted, or one or more residues in the Fc amino acid sequence may be removed. Deletion variants therefore include all fragments of an Fc polypeptide sequence. In substitution variants of Fc, one or more amino acid residues in the Fc polypeptide are removed and replaced by another residue. In one aspect, the substitutions are conservative in nature. However, non-conservative substitutions are also within the scope of the invention. For example, cysteine residues can be deleted or substituted with other amino acids to inhibit disulfide bridges in some or all of the Fc sequences. If the protein has one or more cysteine residues, each of the cysteine residues may be removed, or one or more of the cysteine residues may be replaced with another amino acid (Ala or Ser, or non-naturally encoded amino acids) may be substituted. As another example, the modification may introduce amino acid substitutions for the following purposes: (1) removal of the Fc receptor binding site, (2) removal of the complement (Clq) binding site, and/or (3) Antibody-dependent-elimination of cell-mediated cytotoxicity (ADCC) sites. Such sites are known in the art (see, eg, the ADCC site of IgG1 [Molecular Immunology, Vol. 29, No. 5, 633-639 (1992)]). Any known substitution is also included within the scope of "Fc" as used herein. Similarly, one or more tyrosine residues may be replaced by phenylalanine residues. In addition, other variant amino acid insertions, deletions (eg, of 1-25 amino acids), and/or substitutions are also contemplated and included within the scope of the invention. Generally, conservative amino acid substitutions are preferred. Mutations may also take the form of mutated amino acids (such as peptidomimetics or D-amino acids).

Also, the Fc sequence may be derivatized. That is, modifications other than insertion, deletion or substitution of amino acid residues may be made. Modifications are preferably covalent in nature and include chemical bonding between polymers, lipids and other organic and inorganic moieties. Derivatives of the invention may be engineered to have increased circulation half-lives. Alternatively, it may be designed to enhance the ability of the polypeptide to target a desired cell, tissue or organ. The salvage receptor binding domain of the complete Fc molecule can also be used as the Fc portion of the compounds of the invention (as described in WO 96/32478 ("Altered Polypeptides with Increased Half-Life")). ). Another series of molecules, referred to herein as Fc, are those described in WO 97/34631 (section "Immunoglobulin-Like Domains with Increased Half-Lives"). The two PCT application publications cited in this paragraph are hereby incorporated by reference.

In one embodiment, anti-CD3 antibody compositions having unnatural amino acids (such as p-propargyloxyphenylalanine) are provided. Also provided are various compositions that include, but are not limited to, p-propargyloxyphenylalanine and further proteins and/or cells. In one aspect, the composition comprising the unnatural amino acid p-propargyloxyphenylalanine further comprises an orthogonal tRNA. The unnatural amino acid may be attached (eg, but not limited to, covalently) to an orthogonal tRNA. Examples of this linkage include (i) covalent attachment to an orthogonal tRNA via an amino-acyl bond; binding, and the like, including but not limited to.

[Measurement of activity of anti-CD3 antibody and affinity of anti-CD3 antibody for antigen or binding target of anti-CD3 antibody]
Activity of anti-CD3 antibodies can be measured by standard in vitro or in vivo assays. For example, a cell or cell line that binds an anti-CD3 antibody (e.g., a cell that contains a naturally occurring anti-CD3 antibody antigen or binding partner, or a cell that recombinantly produces an anti-CD3 antibody antigen or binding partner, such as ) can be used to assess binding of anti-CD3 antibodies. For non-PEGylated antigen-binding polypeptides or PEGylated antigen-binding polypeptides having unnatural amino acids, the affinity of the anti-CD3 antibody for the antigen or binding target of the anti-CD3 antibody is technology (eg, BIAcore™ biosensor (Pharmacia), etc.).

Regardless of the method employed to generate the anti-CD3 antibody, the anti-CD3 antibody is subjected to assays to assess biological activity. If appropriate, a tritiated thymidine assay may be performed to confirm the extent of cell division. However, other biological assays may be used to confirm the desired activity. For example, biological assays that measure the ability to inhibit biological activity (such as enzymatic, proliferative or metabolic activity) of an antigen also demonstrate activity of anti-CD3 antibodies. Other in vitro assays may be used to confirm biological activity. In general, when appropriate to the biological activity of the antigen, the bioactivity test should give an assay for the desired result. This analysis includes, for example, increased or decreased biological activity (compared to unmodified anti-CD3 antibody), altered biological activity (compared to unmodified anti-CD3 antibody), receptor affinity assay. , conformational or structural changes, or serum half-life analysis.

The compilation of the above descriptions of assay methods is not to be undue burdensome. Those skilled in the art will also be aware of other assays useful for testing the desired end result.

[Measurement of Efficacy, In Vivo Functional Half-Life, and Pharmacokinetic Parameters]
An important aspect of the present invention is the extended biological half-life obtained by constructing anti-CD3 antibodies, with or without conjugation of anti-CD3 antibodies with water-soluble polymer moieties. is. The rapid decline in serum concentrations of anti-CD3 antibodies has made assessment of biological responses to treatment with conjugated or unconjugated anti-CD3 antibodies and variants thereof important. Preferably, the conjugated or unconjugated anti-CD3 antibodies and variants thereof of the invention have prolonged serum half-life even after intravenous administration. This allows measurement by, for example, ELISA methods or primary screening assays. In vivo biological half-life measurements are performed by methods described herein.

Pharmacokinetic parameters of antigen-binding polypeptides having a non-naturally encoded amino acid can be evaluated in normal Sprague-Dawley rats (male) (N=5 animals per treatment group). Animals are given a single intravenous dose of 25 μg per animal or a single 50 μg subcutaneous dose per animal. Then, about 5 to 7 blood samples are collected according to the passage of time determined in advance. This time course typically extends to about 6 hours for antigen-binding polypeptides that are not conjugated to water-soluble polymers and have non-naturally encoded amino acids. On the other hand, antigen-binding polypeptides conjugated to water-soluble polymers and having non-naturally encoded amino acids usually extend to about 4 days. Pharmacokinetic data for anti-CD3 antibodies have been well studied in several species and can be directly compared to data obtained for anti-CD3 antibodies with non-naturally encoded amino acids.

The specific activity of anti-CD3 antibodies of the present invention can be measured by various known assays. The biological activity of an anti-CD3 antibody mutein or fragment thereof according to the invention, whether obtained or purified, can be determined by methods described or incorporated herein or by a person skilled in the art. can be tested by methods known to

ADMINISTRATION AND PHARMACEUTICAL COMPOSITIONS
Polypeptides or proteins of the invention (including, but not limited to, anti-CD3 antibodies, synthases, proteins, etc. having one or more unnatural amino acids) are optionally employed for therapeutic use. (e.g., but not limited to, in combination with a suitable pharmaceutical carrier). Such compositions comprise, for example, a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and/or combinations thereof. The formulation is made to suit the mode of administration. Generally, methods of administering proteins are well known in the art and can be applied to administration of the polypeptides of the invention.

Therapeutic compositions comprising one or more of the polypeptides of the invention are optionally tested in one or more in vitro and/or in vivo animal models of appropriate diseases to determine efficacy and tissue effects. Metabolism may be checked and dosage may be assessed. This test follows methods well known in the art. Specifically, dosages can be initially determined by activity, stability, or other suitable measures of the natural amino acid homologues of the unnatural amino acids herein (i.e., comparative assays) (e.g., one There is a comparison between anti-CD3 antibodies modified to have unnatural amino acids and anti-CD3 antibodies composed of natural amino acids, but not limited thereto).

Administration may be by any route commonly used for introduction such that the molecule ultimately comes into contact with blood or tissue cells. The non-natural amino acid polypeptides of the invention are administered in any suitable manner, optionally with one or more pharmaceutically acceptable carriers. Suitable methods of administering the polypeptides of the present invention to patients are available. Also, certain routes are usually more rapid-acting and may produce more effective effects or responses than others (although more than one route may be used to administer a particular composition). be able to).

Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and the particular method used to administer the composition. Therefore, there are many different types of suitable formulations of pharmaceutical compositions of the present invention.

A polypeptide composition can be administered by a variety of routes. Such routes include, but are not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual or rectal administration. Compositions containing non-natural amino acid polypeptides (modified or unmodified) can also be administered using liposomes. Such routes of administration and suitable formulations are well known to those skilled in the art.

Anti-CD3 antibodies with unnatural amino acids, alone or in combination with other suitable ingredients, can also be made into aerosol formulations (ie, can be "nebulized") to be administered by inhalation. Aerosol formulations can be placed into pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration (e.g., intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes) include aqueous or non-aqueous, isotonic Sterile injectable solutions are included. This infusion solution contains antioxidants, buffers, bacteriostats, and solutes that keep the formulation isotonic with the blood of the intended patient, as well as aqueous and non-aqueous sterile suspensions (suspending agents, solubilizing agents, It may contain thickeners, stabilizers, preservatives). The packaged anti-CD3 antibody formulation may be enclosed in a single dose or multiple doses in a sealed container (ampule, vial, etc.).

Parenteral and intravenous administration are preferred methods of administration. In particular, routes of administration already used in therapy with natural amino acid homologues (e.g., EPO, GH, anti-CD3 antibodies, G-CSF, GM-CSF, IFN, interleukins, antibodies, and/or any other pharmaceutical routes of administration typically used for proteins delivered to, but not limited to, the preferred routes of administration for the polypeptides of the invention, in conjunction with the dosage form used with that route of administration, and Give the dosage form.

In the context of the present invention, the dosage administered to a patient is that amount sufficient to induce a beneficial therapeutic response in that patient over the long term. Alternatively, depending on the application, it may be an amount that prevents infection by pathogens, or an amount that inhibits other suitable activities (but is not limited to these). Dosage may depend on (i) efficacy of the particular vector or formulation, (ii) activity, stability or serum half-life of the non-natural amino acid polypeptide employed, (iii) patient condition, (iv) patient's Determined according to body weight or body surface area. The single dose will also depend on the lifestyle and constitution of the particular patient and the extent of any undesirable side effects associated with administration of the particular vector, formulation, and the like.

In determining the effective amount of vector or formulation to administer in the treatment or prevention of disease (including but not limited to cancer, genetic disorders, diabetes, AIDS), circulating plasma levels, formulation toxicity, disease progression, and/or (if relevant) the production of anti-non-natural amino acid polypeptide antibody are evaluated by a physician.

For example, dosages administered to a 70 kg patient will generally be within a range that is equivalent to dosages of currently used therapeutic proteins, adjusted for altered activity or serum half-life of the composition. . The vectors of the invention can supplement treatment of the condition by any known therapy. Such known therapies include administration of antibodies, administration of vaccines, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers, and the like.

Administration of the formulations of the present invention may be administered at a pace determined by the LD-50 or ED-50 of the formulation and/or determined by observing any side effects of the unnatural amino acid at various concentrations. Administer at an appropriate pace. In the latter case, this observation may be applied to, but not limited to, the patient's weight and general health. Administration may be completed in a single dose or in divided doses to complete administration.

Administer appropriate doses of aspirin, ibuprofen, acetaminophen, or other antipyretic analgesics if a patient being injected with the formulation complains of fever, chills, or myalgia. Patients who exhibit reactions to infusions such as fever, myalgia and chills are premedicated with either aspirin, acetaminophen or diphenhydramine such as, but not limited to, 30 minutes prior to further infusions. Use meperidine for more severe chills and myalgia that do not respond rapidly to antipyretics and antihistamines. Depending on the severity of the reaction, slow or stop the cell infusion.

The human antigen-binding polypeptides of the present invention can be administered directly to a mammalian subject. Administration is by any route commonly used for introducing anti-CD3 antibodies to a subject. Anti-CD3 antibody compositions according to embodiments of the present invention may be administered orally, rectally, topically, inhaled (including but not limited to aerosols), buccal (including sublingual), but not limited to), intravaginal administration, parenteral administration (subcutaneous administration, intramuscular administration, intradermal administration, intraarticular administration, intrapleural administration, intraperitoneal administration, intracerebral administration, intraarterial administration, intravenous administration, etc.) including but not limited to), topical administration (ie, administration to skin and mucosal surfaces, including administration to airway surfaces), and transdermal administration. However, the optimum route in any given case will depend on the nature and severity of the condition to be treated. Administration can be local or systemic. Formulations of compounds can be presented in unit dose or multi-dose quantities in sealed containers, such as ampoules and vials. The anti-CD3 antibodies of the present invention can be prepared as a mixture with a pharmaceutically acceptable carrier in a single dose injectable form (such as a solution, suspension or emulsion, but but not limited to these). Alternatively, the anti-CD3 antibody of the present invention may be administered by continuous infusion (e.g., using a small pump such as an osmotic pump, but not limited to this), or by a single bolus administration, It may also be administered as a sustained release depot.

Formulations suitable for administration include aqueous or non-aqueous solutions (sterile isotonic solutions) that contain antioxidants, buffers, bacteriostats, and solutes that keep the formulation isotonic. can be done. Aqueous or non-aqueous sterile suspensions are also suitable formulations for administration and may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives. Solutions and suspensions can be prepared from sterile powders, sterile granules, and tablets of the kind previously described.

A pharmaceutical composition of the invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and the particular method used to administer the composition. Suitable formulations (optionally including pharmaceutically acceptable carriers, excipients or stabilizers) of the pharmaceutical compositions of the invention, therefore, are of a wide variety (e.g., [Remington's Pharmaceutical Sciences, ^17th ed. (1985)].

Suitable carriers include: buffers (phosphates, borates, HEPES, citrates and other organic acids); antioxidants (such as ascorbic acid); low molecular weight polypeptides (about proteins (such as serum albumin, gelatin or immunoglobulins); hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates. hydrogen (such as glucose, mannose or dextrin); chelating agents (such as EDTA); divalent metal ions (such as zinc, cobalt or copper); sugar alcohols (such as mannitol or sorbitol); salt-forming counterions (such as sodium); and/or non-ionic surfactants such as Tween™, Pluronics™ or PEG.

Anti-CD3 antibodies of the invention (including antibodies linked to water-soluble polymers such as PEG) can be administered as sustained release systems (or partially as sustained release systems). Sustained-release compositions include (but are not limited to) semipermeable polymer-based shaped articles such as, but not limited to, films or microcapsules. Sustained-release vehicles include biocompatible materials such as: poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 ( 1981); Langer, Chem. Tech., 12: 98-105 (1982)); ethylene-vinyl acetate (Langer et al., supra); or poly-D-(-)-3-hydroxybutyric acid (EP 133, 988); polylactide (polylactic acid) (U.S. Pat. No. 3,773,919, EP 58,481); polyglycolide (polymer of glycolic acid); polylactide-co-glycolide anhydride polymer (lactic acid and glycolic acid copolymer); copolymer of L-glutamic acid and γ-ethyl-L-glutamate (U. Sidman et al., Biopolymers, 22, 547-556 (1983)); poly(ortho)ester; polypeptide; hyaluronic acid; collagen; chondroitin sulfate; carboxylic acids; fatty acids; Sustained-release compositions may also include liposome-encapsulated compounds. Liposomes containing the compounds are prepared by methods known per se. DE 3, 218, 121, [Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985)], [Hwang et al., Proc. 4030-4034 (1980)], EP 52,322, EP 36,676, EP 88,046, EP 143,949, EP 142,641, Japanese Patent Application No. 83-118008, U.S. Pat. See 4,544,545, EP 102,324 (all publications and patent documents cited are incorporated herein by reference).

Liposome-encapsulated anti-CD3 antibodies can be prepared, for example, by methods described in DE 3, 218, 121, [Epstein et al., Proc. Natl. Acad. -3692 (1985)], [Hwang et al., Proc. Natl. Acad. Sci. , 641, Japanese Patent Application No. 83-118008, US Pat. The composition and size of liposomes are well known and can also be readily determined empirically by one skilled in the art. Some examples of liposomes are described, for example, in [Park JW, et al., Proc. Natl. Acad. Sci. (eds): Medical Applications of Liposomes (1998)], [Drummond DC, et al., Liposomal drug delivery systems for cancer therapy, in Teicher B (ed): Cancer Drug Discovery and Development (2002)], [Park JW, et al., Clin. Cancer Res. 8:1172-1181 (2002)]; [Nielsen UB, et al., Biochim. Biophys. Acta 1591(1-3):109-118 (2002); al., Cancer Res. 63: 3154-3161 (2003)] (all publications and patents cited are incorporated herein by reference).

With respect to the present invention, the dosage administered to a patient should be sufficient to induce a beneficial response in the subject over an extended period of time. When the anti-CD3 antibodies of the invention are administered parenterally, the total pharmaceutically effective amount per dose is usually about 0.01 μg/kg/day to about 100 μg/kg, or about 0.01 μg/kg/day to about 100 μg/kg. 05 mg/kg to about 1 mg/kg (per patient body weight). However, dosage is subject to therapeutic judgment. Dosing frequency is also subject to therapeutic judgment. It may be administered more or less frequently than commercial anti-CD3 antibody products approved for use in humans. The PEGylated antigen-binding polypeptides of the invention can generally be administered by any of the routes of administration described above.

[Therapeutic use of the antigen-binding polypeptide of the present invention]
The anti-CD3 antibody polypeptides of the invention are useful in treating a wide variety of disorders. Pharmaceutical compositions containing anti-CD3 antibodies of effective strength when administered by various means to human patients suffering from disorders that may benefit from the use of anti-CD3 antibody agonists or antagonists. general compositions can be formulated. Effects from anti-CD3 antibody agonists or antagonists include, but are not limited to, anti-proliferative, anti-inflammatory, anti-viral, and the like. This effect may be on the condition or disease itself or on a portion of the condition or disease. The average amount of anti-CD3 antibody can vary. Among other things, it should be varied based on the recommendations and prescriptions of a qualified physician. The exact amount of anti-CD3 antibody is a matter of preference, given such factors as the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The invention also provides for the administration of therapeutically effective amounts of other active ingredients, such as anti-cancer chemotherapeutic agents. Dosages can be readily determined by those of ordinary skill in the art based on treatment with an anti-CD3 antibody.

The following examples are offered for illustration and do not limit the claimed invention.

[Example 1]
This example describes one of many possible criteria for selecting a suitable site for incorporation of a non-naturally encoded amino acid into an anti-CD3 antibody.

Demonstrated in this example is a method for choosing a suitable site in an antigen-binding polypeptide to introduce a non-naturally encoded amino acid. The three-dimensional structure composed of two anti-CD3 antibody molecules or the secondary, tertiary or quaternary structure of the anti-CD3 antibody may be used to introduce one or more non-naturally encoded amino acids. Determine a suitable location.

The following criteria are used to evaluate each position of the anti-CD3 antibody for introducing a non-naturally encoded amino acid.
That is, the residue is
(a) should not interfere with binding of the anti-CD3 antibody based on structural analysis of the three-dimensional structure, nor should it interfere with the secondary, tertiary or quaternary structure of the anti-CD3 antibody;
(b) not be affected by scanning mutagenesis with alanine or homologues;
(c) must be exposed to the surface and have minimal van der Waals or hydrogen bonding interactions with surrounding residues;
(d) may be at one or more exposed surfaces of the anti-CD3 antibody;
(e) may be one or more anti-CD3 antibody sites proximal to a second anti-CD3 antibody, or other molecule or fragment thereof;
(f) must be either deleted or variable in the anti-CD3 antibody variant;
(g) may be conservatively varied as a result of substitution with non-naturally encoded amino acids;
(h) may modulate the configuration of the anti-CD3 antibody itself, or of dimers or multimers comprising one or more anti-CD3 antibodies, by desirably altering the flexibility or rigidity of the overall structure;
(i) may be in either highly flexible regions or structurally rigid regions;
(j) may or may not be in a complementarity determining region (CDR);

Further calculations are also performed on the anti-CD3 antibody molecule with the Cx program (Pintar et al. Bioinformatics, 18, pp 980) to assess the degree of protrusion for each atom in the protein. Consequently, in some embodiments, without limitation, one or more sites in the anti-CD3 antibody are substituted with non-naturally encoded amino acids.

[Example 2]
This example details the cloning and expression in E. coli of anti-CD3 antibodies with non-naturally encoded amino acids.

A transduction system comprising an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl-tRNA synthetase (O-RS) is used to express anti-CD3 antibodies with non-naturally encoded amino acids. The O-RS preferentially aminoacylates non-naturally encoded amino acids and O-tRNAs. The translation system then inserts the non-naturally encoded amino acid into the anti-CD3 antibody corresponding to the selected codon encoded.

Transform E. coli with a plasmid containing the modified anti-CD3 antibody gene and an orthogonal aminoacyl-tRNA synthetase/tRNA pair (specific for the desired non-naturally encoded amino acid). This allows site-specific incorporation of non-naturally encoded amino acids into anti-CD3 antibodies. Engineered anti-CD3 antibodies are produced with high accuracy and efficiency by culturing the transformed E. coli at 37° C. in medium containing 0.01-100 mM of the specific non-naturally encoded amino acid. express. An anti-CD3 antibody having a non-naturally encoded amino acid tagged with a His tag is produced in E. coli host cells. The product is obtained as inclusion bodies or as aggregates. Aggregates are solubilized and affinity purified in a denaturing environment (6M guanidine hydrochloride). The protein is refolded by a dialysis method (in 50 mM TRIS-HCl (pH 8.0), 40 μM CuSO ₄ and 2% (w/v) sarkosyl at 4° C. overnight). The resulting material is then dialyzed in 20 mM TRIS-HCl (pH 8.0), 100 mM NaCl and 2 mM CaCl ₂ after which the His-tag is removed ([Boissel et al., (1993) 268:15983- 93]). Methods for purifying anti-CD3 antibodies are well known and can be confirmed by SDS-PAGE, Western blot analysis, electrospray ionization-ion trap mass spectrometry, or the like.

[Expression/Repression]
(Suppression by para-acetylphenylalanine (pAcF))
E. coli amber mutations were suppressed according to known standard protocols. Briefly, the following manipulations were performed to suppress antibody fragments (scFv and Fab) in the periplasm of E. coli. First, E. coli host cells were transformed with an expression vector construct (a plasmid encoding an orthogonal tyrosyl-tRNA synthetase (MjTyrRS) from M. jannaschii). After overnight, the bacterial medium was diluted 1:100 in a flask containing LB (Luria-Bertani) medium or Superbroth with shaking. Then, it was cultured at 37° C. until the OD reached about 0.8. Fab and scFv expression was induced. Para-acetylphenylalanine (pAcF) was also added to a final concentration of 4 mM to suppress amber codons. The medium was incubated overnight at 25°C.

(suppressed by aa9.2)
The amber mutation was suppressed by a pAcF derivative (aa9.2) in a manner similar to pAcF, except that an orthogonal tyrosyl-tRNA synthetase from M. jannaschii, which is specific for this amino acid, was used. Upon induction, it was suppressed by adding 4 mM aa9.2.

[Protein extraction and purification]
Cells were harvested by centrifugation and resuspended in periplasmic release buffer (50 mM _NaPO4 , 20% sucrose, 1 mM EDTA; pH 8.0) supplemented with 100 μg/mL lysozyme. It was then incubated on ice for 30 minutes. After centrifugation, antibody fragments in the supernatant were immobilized on ProBind beads (Invitrogen; Carlsbad, Calif.) utilizing the His-tag of the antibody fragments. After washing the beads extensively with binding buffer, bound fragments were eluted from the beads with 0.5 M imidazole. Purified fragments were dialyzed in storage buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 5% sucrose; pH 7.8). For small-scale analysis of scFv fragments expressed in the cytoplasm, E. coli in 15 mL of medium were harvested by centrifugation. They were then resuspended in 1 mL lysis buffer (B-PER, Pierce Biotechnology; Rockford, Ill.) supplemented with 10 μg/mL DNAse. The mixture was incubated at 37° C. for 30 minutes, diluted 1× with Protein Loading Buffer (Invitrogen; Carlsbad, Calif.) and analyzed by SDS-PAGE.

[Example 3]
This example details the introduction of an amino acid having a carbonyl. The subsequent reaction with aminooxy-containing PEG will also be described in detail.

This example demonstrates a method for making an antigen-binding polypeptide that incorporates an amino acid with a non-naturally encoded ketone. This ketone-bearing amino acid is then reacted with an aminooxy-bearing PEG (molecular weight: about 5,000). Each of the residues identified by the criteria in Example 1 is individually replaced with a non-naturally encoded amino acid having the structure below.

After modification, the anti-CD3 antibody variants with carbonyl-bearing amino acids are reacted with aminooxy-bearing PEG derivatives (structure below).
R-PEG(N)-O-( _CH2 ) _n -O- _NH2 .

wherein R is methyl, n is 3 and the molecular weight of N is about 5,000. Anti-CD3 antibodies bearing p-acetylphenylalanine were purified and added to (i) 25 mM MES (Sigma Chemical, St. Louis, MO; pH 6.0), (ii) 25 mM Hepes (Sigma Chemical, St. Louis , MO; pH 7.0) or (iii) 10 mM sodium acetate (Sigma Chemical, St. Louis, MO; pH 4.5) (10 mg/mL concentration). This solution is reacted with a 10- to 100-fold excess of PEG with aminooxy. And then stir at room temperature for 10-16 hours (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). The PEG-anti-CD3 antibody is then diluted in an appropriate buffer and immediately purified and analyzed.

[Example 4]
Conjugation with PEG, having a hydroxylamine group linked to PEG via an amide bond.

A PEG reagent having the structure below is coupled with an amino acid bearing a non-naturally encoded ketone by the method described in Example 3.
R-PEG(N)-O-( _CH2 ) ₂ -NH-C(O)( _CH2 ) _n -O- _NH2 .

wherein R is methyl, n is 4 and the molecular weight of N is about 20,000. The reaction, purification and analysis conditions are as described in Example 3.

[Example 5]
This example details the introduction of two different non-naturally encoded amino acids into an anti-CD3 antibody.

This example demonstrates a method for producing an antigen-binding polypeptide that incorporates two non-naturally encoded amino acids with ketone function. The positions of integration are identified according to Example 1, with X ^* representing non-naturally encoded amino acids. Antigen-binding polypeptides are produced as described in Examples 1 and 2, except that the suppression codon is introduced at two different sites in the nucleic acid.

[Example 6]
This example details the conjugation of an antigen-binding polypeptide with a hydrazide-bearing PEG. In addition, surgery is performed for the subsequent in situ reduction.

Antigen-binding polypeptides incorporating carbonyl-bearing amino acids are made according to the procedures described in Examples 2 and 3. After modification, the hydrazide-bearing PEG (structure below) is conjugated to the anti-CD3 antibody.
R-PEG(N)-O-( _CH2 ) ₂ -NH-C(O)( _CH2 ) _n -X-NH- _NH2 .

wherein R is methyl, n is 2, N has a molecular weight of 10,000, and X is a carbonyl group (C=O). Anti-CD3 antibodies bearing p-acetylphenylalanine were purified and added to (i) 25 mM MES (Sigma Chemical, St. Louis, MO; pH 6.0), (ii) 25 mM Hepes (Sigma Chemical, St. Louis , MO; pH 7.0) or (iii) 10 mM sodium acetate (Sigma Chemical, St. Louis, MO; pH 4.5) (concentration 0.1-10 mg/mL). This solution is reacted with a 1- to 100-fold excess of PEG bearing a hydrazide. A 1 M NaCNBH ₃ stock (Sigma Chemical, St. Louis, Mo.; final concentration 10-50 mM; dissolved in H ₂ O) is then added to reduce the corresponding hydrazone in situ. The reaction is allowed to proceed for 18-24 hours at 4° C. to room temperature in the dark. The reaction is stopped by the addition of 1M Tris (Sigma Chemical, St. Louis, Mo.; pH approximately 7.6; final concentration of Tris 50 mM or diluted with an appropriate buffer) and immediately purified.

[Example 7]
This example details the introduction of alkyne-bearing amino acids into an anti-CD3 antibody. It also details derivatization with mPEG-azide.

Any residue identified according to Example 1 is substituted with the non-naturally encoded amino acid below.

Anti-CD3 antibodies bearing propargyl tyrosine are purified and dissolved in PB buffer (100 mM sodium phosphate, 0.15 M NaCl; pH 8) (concentration 0.1-10 mg/mL). A 10- to 1000-fold excess of PEG bearing an azide is added to the reaction mixture. A catalytic amount of CuSO ₄ and copper wire are then added to the reaction mixture. After incubating the mixture (for example, but not limited to, at room temperature or 37° C. for about 4 hours, or at 4° C. overnight), H ₂ O is added and the mixture is filtered through a dialysis membrane. . Samples can be analyzed by procedures similar to, but not limited to, those described in Example 3.

The structure of PEG in this example is as follows.
R-PEG(N)-O-( _CH2 ) _2- NH-C(O)( _CH2 ) _n - _N3 .

wherein R is methyl, n is 4 and the molecular weight of N is 10,000.

[Example 8]
This example details the replacement of large hydrophobic amino acids in anti-CD3 antibodies with propargyl tyrosine.

Phe, Trp or Tyr residues present in the sequence of the anti-CD3 antibody are replaced with the following non-naturally encoded amino acids as described in Example 7.

After modification, PEG is added to the anti-CD3 antibody variants having amino acids with alkynes. The structure of this PEG is shown below, and the coupling procedure is according to that described in Example 7.
Me-PEG(N)-O-( _CH2 ) ₂ - _N3 .

thereby (i) having a non-naturally encoded amino acid that is approximately equivalent to a naturally occurring large hydrophobic amino acid, and (ii) being modified with a PEG derivative at discrete sites in the polypeptide. , anti-CD3 antibody variants are generated.

[Example 9]
This example details the generation of anti-CD3 antibody homodimers, heterodimers, homomultimers or heteromultimers separated by one or more PEG linkers.

An alkyne-bearing anti-CD3 antibody variant produced according to Example 7 is reacted with a bifunctional PEG derivative (structure below).
N ₃ —(CH ₂ ) _n —C(O)—NH—(CH ₂ ) ₂ —O—PEG(N)—O—(CH ₂ ) ₂ —NH—C(O)—(CH ₂ ) _n — _N3 .

In the formula, n is 4 and the average molecular weight of PEG is about 5,000. The reaction produces homodimers of the corresponding anti-CD3 antibodies. In this dimer, two anti-CD3 antibody molecules are physically separated by PEG. By similar techniques, antigen-binding polypeptides can be coupled to one or more other polypeptides to form heterodimers, homomultimers or heteromultimers. Coupling, purification and analysis are performed as in Examples 7 and 3.

[Example 10]
This example describes methods for measuring the in vitro and in vivo activity of anti-CD3 antibodies having non-naturally encoded amino acids and pegylated anti-CD3 antibodies.

[Cell binding assay]
Incubate cells (3×10 ⁶ ) in PBS/1% BSA (100 μL) in pairs. Incubation was performed in the presence or absence of (i) various concentrations of unlabeled anti-CD3 antibody, anti-CD3 antibody or negative control (volume: 10 μL) and (ii) ¹²⁵ I-anti-CD3 antibody ( 100,000 cpm or 1 ng) at 0° C. for 90 minutes (total volume: 120 μL). Cells are then resuspended and layered over ice-cold FCS (200 μL) in a 350 μL plastic centrifuge tube and centrifuged (1000 g; 1 min). The end of the tube is cut to collect the pellet, and the pellet and supernatant are measured separately using a gamma counter (Packard).

Specific binding (cpm) was calculated from total binding in the absence of competitor (average of one of the duplicates) to binding in the presence of a 100-fold excess of unlabeled anti-CD3 antibody (non-specific specific binding; cpm). Non-specific binding is determined for each cell type used. Experiments should be performed with the same ¹²⁵ I-anti-CD3 antibody preparation on different dates and the results of the trials should show internal consistency. Binding is inhibited in a dose-dependent manner of unlabeled native anti-CD3 antibody or anti-CD3 antibody, but not by the negative control. The ability of the anti-CD3 antibody to compete with the binding of the native ¹²⁵ I-anti-CD3 antibody suggests that the receptor recognizes both forms equally well.

[In vivo study of PEGylated anti-CD3 antibody]
PEG-anti-CD3 antibody, unmodified anti-CD3 antibody and buffer solution are administered to mice or rats. It is expected that the PEGylated anti-CD3 antibodies of the present invention will exhibit improved activity and an extended half-life compared to unmodified anti-CD3 antibodies.

[Measurement of in vivo half-life of conjugated anti-CD3 antibody, non-conjugated anti-CD3 antibody and variants thereof]
Male Sprague Dawley rats (approximately 7 weeks old) are used. On the day of dosing, each animal is weighed. Three rats each are injected intravenously via the tail vein with 100 μg unconjugated or conjugated anti-CD3 antibody samples per kg body weight. At 1 minute, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours after injection, 500 μL of blood is taken from each rat while anesthetized with CO ₂ . Blood samples are stored at room temperature for 1.5 hours, after which serum is separated by centrifugation (4° C., 18,000×g for 5 minutes). Serum samples are stored at -80°C until the day of analysis. After thawing the samples on ice, the amount of active anti-CD3 antibodies in serum samples is determined by an anti-CD3 antibody in vitro activity assay.

[Example 11]
Human safety and/or efficacy clinical trials of pegylated anti-CD3 antibodies with non-naturally encoded amino acids.

[the purpose]
To compare the safety and pharmacokinetics of subcutaneously administered pegylated recombinant human anti-CD3 antibodies with non-naturally encoded amino acids compared to commercial products specific for the same target antigen. . Examples of commercial products are Herceptin, Bexxar, Campath, CEA-Scan, Enbrel, Erbitux, Humira, Myoscint, Prostascint, Raptiva, Remicade, ReoPro, Rituxan, Siμlect, Synagis, Verluma, Xolair, Zenapax, Zevalin or Avastin. (all registered trademarks).

[patient]
Eighteen healthy volunteers are enrolled in the study (age 20-40 years; body weight 60-90 kg). Subjects have no clinically significant laboratory abnormalities in hematology or serum chemistry and are negative for both urine toxicity screen, HIV screen and hepatitis B surface antigen. Subjects must not have the following signs: hypertension; history of any primary hematologic disease; serious liver, renal, cardiovascular, gastrointestinal, renal-urogenital, metabolic, neurological History of disease; history of anemia or seizure disorder; known susceptibility to bacterial or mammalian-derived products, PEG or human serum albumin; habitual and heavy consumption of caffeinated beverages; participation in any other clinical trial; Or participation in a blood transfusion or blood donation within 30 days before the start of the study; exposure to anti-CD3 antibodies within 3 months before the start of the study; disease within 7 days before the start of the study; physical examination before the study, or start of the study Serious abnormalities in clinical trial assessments within the previous 14 days. All subjects can be evaluated for safety. Blood for pharmacokinetic analysis will also be drawn on schedule. The entire study is conducted under institutional review board approval and patient consent.

[Test design]
This study is a Phase I, single-center, open-label, randomized, two-period crossover study in healthy male participants. Eighteen subjects are randomly assigned to one of two treatment sequence groups (9 subjects per group). Anti-CD3 antibody is administered by subcutaneous bolus injection into the upper thigh in two separate dosing periods. Administered are equivalent doses of a pegylated anti-CD3 antibody having a non-naturally encoded amino acid and the selected commercial product. Dosage and frequency of administration of commercially available products should be in accordance with the instructions in the package insert. Additional doses, dosing frequencies or other desired parameters using commercial products may be added to the study by including additional subject groups. Each dosing period is separated by a 14-day washout period. For each of the two dosing periods, subjects are confined to the study facility for a minimum of 12 hours prior to dosing and 72 hours after dosing. However, there are no restrictions during the administration period. Additional subject groups may be added if additional doses, dosing frequencies or other parameters are tested for the PEGylated anti-CD3 antibody. An experimental formulation of an anti-CD3 antibody is a PEGylated anti-CD3 antibody with non-naturally encoded amino acids.

[Blood sampling]
Serial blood is drawn by direct venous puncture before and after administration of anti-CD3 antibody. To examine anti-CD3 antibody concentrations in serum, (i) at approximately 30, 20 and 10 minutes prior to dosing (three baseline samples) and (ii) approximately 30 minutes, 1 hour after dosing, Venous blood samples (5 mL) are taken at 2, 5, 8, 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Divide each serum sample into two halves. All serum samples are stored at -20°C. Serum samples are shipped on dry ice. Fasting laboratory tests (blood, serum chemistries, and urinalysis) are performed on Day 1 just prior to first dose, Day 4 morning, Day 16 just prior to dose, and Day 19 morning.

[Method of bioanalysis]
Anti-CD3 antibody concentration in serum is determined by radioimmunoassay (RA) method or ELISA kit method.

[Safety judgment]
Vital signs are recorded immediately prior to each dose (Days 1 and 16) and 6, 24, 48 and 72 hours after each dose. Safety determinations are based on the occurrence and type of adverse events and changes from baseline in clinical laboratory studies. In addition, changes from pre-study in vital sign (including blood pressure) measurements and physical examination results will be assessed.

[Data analysis]
Post-dose serum concentration values are calibrated to the pre-dose baseline anti-CD3 antibody concentration by subtracting the mean baseline anti-CD3 antibody concentration from each of the post-dose values. Mean baseline anti-CD3 antibody concentrations are determined from the mean anti-CD3 antibody levels from 3 samples taken at 30, 20 and 10 minutes prior to dosing. If the pre-dose serum anti-CD3 antibody concentration is below the level of assay quantitation, it is not included in the calculation of the mean. Pharmacokinetic parameters are determined from serum concentration data calibrated at baseline anti-CD3 antibody concentration. Pharmacokinetic parameters are calculated by a model-independent method using the latest version of BIOAVL software on a Digital Equipment Corporation VAX 8600 computer system. The following pharmacokinetic parameters are determined: peak serum concentration (C _max ); time to reach peak serum concentration (t _max ); time 0 to last blood sampling time (AUC _0-72) calculated by the linear trapezoidal rule. area under the concentration-time curve (AUC) in ); elimination half-life (t _1/2 ), calculated from the elimination rate constant. Elimination rate constants are estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time plot. Mean, standard deviation (SD) and coefficient of variation (CV) of pharmacokinetic parameters are calculated for each treatment. Calculate the ratio of the mean values of the parameters (preserved/not preserved).

[Safety results]
The incidence of adverse events is evenly distributed among the treatment groups. There were no clinically significant changes from baseline, pre-study laboratory tests or blood pressure. Also, there were no notable changes from pre-study physical examination results and vital sign measurements. The safety profile should be similar between the two treatment groups.

[Pharmacokinetic results]
Mean-time profiles of serum anti-CD3 antibody concentrations after a single dose of one or more commercial products specific for the same target antigen (relative to baseline anti-CD3 antibody levels) for all 18 subjects (before calibration) were compared to those of pegylated anti-CD3 antibodies with non-naturally encoded amino acids measured at the same time points. All subjects' predose baseline anti-CD3 antibody concentrations must fall within the normal physiological range. Pharmacokinetic parameters are determined by serum data obtained from mean pre-dose baseline anti-CD3 antibody concentrations. C _max and t _max are then determined. The average t _max of the selected clinical comparator(s) is significantly shorter than the t _max of the pegylated anti-CD3 antibody having the non-naturally encoded amino acid. The elimination half-life values of the commercial anti-CD3 antibody products tested are significantly shorter compared to the elimination half-life of PEGylated anti-CD3 antibodies with non-naturally encoded amino acids.

Although this study was conducted in healthy male subjects, similar absorption profiles and safety profiles are expected in other patient populations (male or female patients with cancer or chronic renal failure, pediatric patients with renal insufficiency, patients scheduled for preoperative self-storage, or patients scheduled for elective surgery).

In conclusion, subcutaneous administration of a single dose of a pegylated anti-CD3 antibody with a non-naturally encoded amino acid is safe and well tolerated in healthy male subjects. Commercial forms of anti-CD3 antibodies and PEGylated anti-CD3 antibodies with non-naturally encoded amino acids showed comparable performance based on comparison of adverse event incidence, clinical laboratory values, vital signs and physical examination results. Has a safety profile. PEGylated anti-CD3 antibodies with non-naturally encoded amino acids could provide significant clinical benefit to patients and healthcare professionals.

[Example 12]
FR ⁺ and FR ⁻ cell lines were used to assess the affinity and specificity of anti-CD3Fab-folate conjugates. Nasopharyngeal carcinoma cell lines (KB) and ovarian cancer cell lines (OV-90, SKOV-3) overexpressed FRs as determined by flow cytometry after culture in folic acid-free medium. . A renal carcinoma cell line (CAKI-1) that does not express FRs and an alveolar basal epithelial cell adenocarcinoma (A549) served as negative controls. Anti-CD3 Fab-folate conjugates were capable of binding KB cells (FR ⁺ ) with similar affinities to antibodies not conjugated to folate. This indicates that conjugation with anti-CD3 Fab results in little loss of binding to the folate receptor. Similarly, anti-CD3 Fab-folate bound to Jurkat cells (CD3 ⁺ ) with similar affinity as anti-CD3. On the other hand, no binding to A549 cells (FR ⁻ ) was observed.

[Example 13]
The activity of anti-CD3 Fab-folate conjugates was then examined by in vitro cytotoxicity assays using KB cells and OV-90 cells (FR ⁺ ). CAKI-1 cells and A549 cells (FR ⁻ ) served as controls. Cells were cultured with activated human peripheral blood mononuclear cells (PMBC) (target cells:effector cells=1:10) and treated with various concentrations of anti-CD3 Fab-folate or anti-CD3 Fab alone. LDH levels released from lysed cells were measured, and CellTiter-Glo and FACS-based injury assays were performed to quantify cytotoxicity. In the presence of PBMCs, anti-CD3 Fab-folate conjugates killed KB and OV-90 cells with _EC50s of 10 pM and 100 pM, respectively (Fig. 2A, Supplementary Fig. 4). However, CAKI-1 cells (Fig. 2A) and A549 cells (Appendix Fig. 4) were unaffected even by 100 nM of the conjugate. Treatment with anti-CD3 Fab alone induced negligible damage in all cell lines examined (Fig. 2A). In addition, SKOV-3 cells (Fig. 2B) or KB cells (Appendix Fig. 5) were incubated with activated PBMCs in folate-free medium for 16 hours in the presence of anti-CD3Fab-folate conjugates (target cells : effector cells=1:10), rosettes were formed. This provides further evidence for targeting by T cells. In contrast, anti-CD3 Fab had no effect on attack by T cells and no aggregation was observed in the absence of PBMCs or in KB cells (Appendix Figure 5).

[Example 14]
Next, we investigated the pharmacokinetics of anti-CD3 Fab-folate conjugates and unconjugated anti-CD3 Fabs in rodents. (i) 1 mg/kg or 5 mg/kg anti-CD3 Fab-folic acid (in PBS) or (ii) 1 mg/kg unconjugated anti-CD3 Fab was administered once by intravenous infusion to 3 rats. Serum collected at regular intervals was then analyzed by ELISA. Both anti-CD3 Fab-folate and the corresponding non-conjugated anti-CD3 Fab variant decreased in serum concentrations at the same rate (serum half-life: 60 min, Figure 3D). Thus, anti-CD3 Fab-folate had similar pharmacokinetics as unconjugated anti-CD3 Fab. This indicates that the PK profile of the conjugate is unaffected by derivatization with folic acid.

[Example 15]
Next, the in vivo efficacy of anti-CD3 Fab-folate bispecific agents was evaluated in a xenograft model using female NOD-SCID mice. Animals were fed a low-folate diet to reduce circulating serum folate levels that could compete with the bispecific drug. KB cells (FR ⁺ ) or A549 cells (FR ⁻ ) were mixed with non-activated human PBMCs (target cells:effector cells=1:100) or activated human PBMCs (target cells:effector cells=1:10). Tumorigenicity was observed when the mixture was injected subcutaneously into mice. Therefore, mice were implanted with KB cells (target cells: effector cells=1:10) mixed with human PBMCs. The same mice were then injected intravenously with 1.5 mg/kg anti-CD3 Fab-folic acid or PBS (starting on the same day as tumor cell implantation and continued daily for 10 days). This dosing regimen was chosen based on the pharmacokinetic properties of anti-CD3 Fab-folate conjugates and previous studies of bispecific ^antibodies10 . A rapid increase in tumor volume was seen in mice treated with PBS (approximately 5-day doubling rate). In contrast, tumors in mice treated with anti-CD3 Fab-folate were barely detectable throughout the study (Fig. 3A). In a parallel study, mice were implanted with KB cells mixed with non-activated human PBMC (target cells:effector cells=1:100). They were then treated intravenously with 1.5 mg/kg anti-CD3 Fab-folic acid or PBS (starting on the same day as tumor cell implantation and continued daily for 10 days). Both groups of mice had reduced tumor volumes during the first 30 days of the study. This is probably due to the high load of PBMCs at the injection site. However, the rate of tumor reduction was faster in mice treated with anti-CD3Fab-folic acid. From study day 35 onwards, tumors in PBS-treated mice gradually increased in volume. In contrast, tumors in mice treated with anti-CD3 Fab-folate decreased to a marginally detectable extent. This indicates that anti-CD3 Fab-folate conjugates eliminate tumors by attacking cytotoxic T cells even in the absence of activated T cells. To examine the in vivo selectivity of anti-CD3 Fab-folate conjugates with respect to FR-expressing tumors, A549 cells (FR ⁻ ) were treated with non-activated human PBMC (target: effector = 1:100). and implanted in mice. Mice were treated in a similar manner (1.5 mg/kg anti-CD3 Fab-folic acid intravenously for 10 consecutive days). As a result, 20 days after treatment, no significant difference was observed from PBS-treated tumors. No body weight loss or overt toxicity was observed in anti-CD3 Fab-folic acid or PBS-treated mice throughout all of the above experiments (Fig. 3C, Appendix Fig. 6B). In addition, histopathological analysis of anti-CD3 Fab-folate-injected and non-injected mice was performed. No T cell infiltration was observed in kidney staining of mice treated with anti-CD3 Fab-folate or PBS. Also, no clear pathological differences in hematoxylin and eosin (H&E) staining images were observed between treated and control groups for other tissues (such as spleen, lung and liver).

[Example 16]
Solid-phase peptide synthesis (SPPS) was performed using a standard peptide synthesizer. All peptides and peptide conjugates were purified using a preparative reverse-phase high performance liquid chromatography (RP-HPLC) apparatus (Waters, xTerra C18 10 μm; 19 x 250 mm). It was then analyzed by analytical RP-HPLC (Waters, X-Bridge C18 5 μm; 3.0×50 mm) and liquid chromatography/mass spectrometry (LC/MS). 1H spectra were obtained using a Varian 400 MHz NMR spectrometer. Samples were run in DMSO-d6/D2O. All 1H signals were reported in ppm with residual solvent or DMSO (2.50 ppm) as reference. Along with the spin coupling constant (Hz), s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet or unresolvable, b: broad line. Protein masses were obtained at the Ambrx mass spectrometry facility (La Jolla, Calif.).

[Example 17]
Construction of expression plasmids, E. coli cell line W3110B60, protein production.

Expression plasmids (AXID) were constructed from pET-20b (+) and pET-24 (+) plasmids (EMD4Biosciences). An amber suppression cassette consisting of a modified M. jannaschiit RNA synthetase (specific for pAcPhe) was cloned into the BamHI site of AXID (type II cloning). The UCHT1 Version.2 Fab CD3 sequence was obtained from a previous publication ^1,2 . This sequence was subcloned into the open reading frame downstream of the phoA promoter in the AXID vector. Lysine 136 (HC-Lysl36) of the heavy chain was QuickChanged to an amber nonsense codon of "TAG" (Stratagene). The wild-type E. coli K-12 W3110 cell line (ATCC) was modified by homologous recombination to have the following genotype ³ :F-IN(rrnD-rrnE)λ-araB::g1 tetA fhuA:: dhfr proSW375R::cat. The temperature-sensitive phenotypic proS present in the W3110B60 genome was complemented by a wild-type, functional copy of proS in this expression plasmid. The vector was transformed into W311B60 cells. The cells were grown in fermentors in synthetic medium (2.1 L) supplemented with kanamycin sulfate (50 μg/mL) at 37° C., pH 7, 480-1200 rpm cascade control. For the production phase, when OD600=30, the pH was changed to 6.8 and synthetic medium was added. Then pAcPhe (100 g/L) dissolved in water was added to the fermentor. Dissolved oxygen was set to remain at 30% and agitation was left at initial conditions (480-1200 rpm). After 24 hours, cells were harvested and UCHT1 was extracted from the cells. Extraction of UCHT1 was performed in TES buffer (0.2M Tris (pH 8.0), 0.5mM EDTA, 0.5M sucrose) at a ratio of 4:1 (v/w) and incubated at 4°C. by giving a shock. The extract was clarified by centrifugation (18000 rpm, 20 min, 4° C.), filtered through a 0.22 micron filter and loaded onto a Protein G column (GE healthcare). The column was washed with 5 bed volumes of PBS (pH 7.4) and protein was eluted with 10 bed volumes of 0.1 M glycine-HCl (pH 3.0). Each fraction was collected in a tube containing 10% 1M Tris-HCl (pH 8). Collected fractions were further purified by cation exchange (column: SP650S (Tosoh Bioscience), buffer A: 0.02 M phosphate (pH 6.0), buffer B: 0.02 M phosphate (pH 6.0) +0.5M NaCl). They were then analyzed by SDS-PAGE gel and mass spectrometry.

[Example 18]
Synthesis of folate-N ¹⁰ (TFA)-Lys linker (1).

Folate-Lys was synthesized by standard solid-phase peptide synthesis starting with H-Lys-(Boc)-2-ClTrt-Resin resin (Peptide international) ⁴ . Briefly, H-Lys-(Boc)-2-ClTrt-Resin resin (500 mg, 0.89 mM) was swollen with DCM (5 mL) followed by further swelling with dimethylformamide (DMF, 5 mL). A solution of Fmoc-L-Glu(OtBu)-OH (1.5 eq), HATU (1.5 eq) and DIPEA (4.0 eq) in DMF (3 mL) was added. Argon was bubbled for 2 hours and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Coupling efficiency was assessed by the Kaiser test. After swelling the resin in DMF (3 mL), a solution of pteroyl acid (1.5 eq), HATU (1.5 eq) and DIPEA (4.0 eq) in DMF (3 mL) was added. Argon was bubbled for 2 hours and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Coupling efficiency was assessed by the Kaiser test. The resin was swelled in DCM (3 mL) and dried under argon. The final compound was cleaved directly from the resin using a cocktail of trifluoroacetic acid (TFA): _H2O :triisopropylsilane (95.5:2.5:2.5) (3 x 5 mL) followed by diethyl ether (300 mL). A crude product was obtained which precipitated in a round-bottomed flask containing After filtration and drying, the crude product was purified using preparative reverse-phase (RP)-HPLC (C18 column, 0% B to 50% B (25 min), buffer A: 0.1 TFA (aq) , buffer B: 0.1 TFA (acetonitrile solution), λ=280 nm). ACN was removed under vacuum and the purified fractions were dry-frozen to give 1 as a pale yellow solid.
^<1> H NMR (DMSO- _d6 / _D2O ): [delta] 1.18 (m, 2H, Pep-H); 1.45 (m, 1H, Pep-H); 1.54 (m, 3H, Pep-H); 1.81 ( 1.95 (m, 1H, Pep-H); 2.11 (m, 2H, Pep-H); 2.88 (m, 2H, Pep-H); 3.94 (m, 1H, Lys- 4.12 (m, 1H, Glu-αH); 4.46 (s, 2H, Pte-H); 6.61 (d, J = 8.5 Hz, 2H, Pte-Ar-H); 7.56 (d, J = 8.5 Hz, 2H, Pte-Ar-H); 8.60 (s, 1H, Pte-Ar-H)
LC-MS (M + H)+: calcd for _C27H30F3N9O8 = 665.6; found = 666.0 g/mol.

[Example 19]
Synthesis of phthalimido-PEG4-COOH.

A solution of triphenylphosphine (TPP, 216.7 mg, 0.826 mmol) and diethyl azodicarboxylate (DEAD, 143.9 mg, 0.826 mmol) in anhydrous THF (0.50 mL) was heated at 0° C. with argon. Under atmosphere, ^t BuOOC-PEG ₄ -OH (200.0 mg, 0.751 mmol) dissolved in anhydrous THF was added. After reacting for 15 minutes with stirring, a solution of N-hydroxyphthalimide (134.8 mg, 0.826 mmol) in anhydrous THF was added over 10 minutes. The reaction mixture was stirred at room temperature under an argon atmosphere for 12 hours. After removing the solvent under vacuum, TFA:triisopropylsilane:water (95:2.5:2.5; 5 mL) was added to the crude product. Then, it was stirred at room temperature for 1 hour to unprotect the tert-butyl group. The final product was purified by flash chromatography (hexane:EtOAc=1:1). The purified fractions were combined and the solvent was evaporated under vacuum to give the final product as an oily liquid.
1HNMR (CDCl3): δ2.63 (m, 2H); 3.59-3.65 (m, 14H); 3.88 (m, 2H); 4.38 (m, 2H); 7.75 (m, 2H), 7.84 (m, 2H)
LC/MS (ESI) (m/z): (M + H ) ⁺ calcd. for C19H25NO9, 411.4, found, 411.4 g/mol.

[Example 20]
Synthesis of phthalimido-PEG4-NHS (2).

HOOC-PEG4-O—N-phthalimide (50 mg, 0.122 mmol), N-hydroxysuccinimide (NHS, 15.4 mg, 0.134 mmol) and 4-dimethylaminopyridine (DMAP, 1.5 mg, 0.0122 mmol). To a solution in anhydrous DCM (0.5 mL) under argon was added ethyl(dimethylaminopropyl)carbodiimide (EDC, 20.8 mg, 0.134 mmol) dissolved in anhydrous DCM (0.5 mL). rice field. The reaction mixture was stirred at room temperature under an argon atmosphere for 4 hours. It was then purified by flash chromatography (hexane:DCM=1:1) to give NHS-PEG4-ON-phthalimide. The purified fractions were combined and the solvent was evaporated under vacuum to give the final product as an oily liquid.
1H NMR: δ 2.90 (t, J=6.4, 2H); 2.75 (bs, 4H); 3.56-3.63 (m, 12H), 3.83-3.87 (m, 4H), 4.37 (t, J=4.5, 2H); 7.75 (m, 2H), 7.83 (m, 2H)
LC/MS (ESI) (m/z): (M + H) ⁺ calcd. for C23H28N2O11, 508.5, found, 509.0 g/mol.

[Example 21]
Synthesis of folate-Lys-PEG4-phthalimide (3).

A solution of NHS-OC-PEG4-ON-phthalimide (21 mg, 0.041 mmol) and folic acid-N ¹⁰ (TFA)-Lys (25 mg, 0.037 mmol) in anhydrous DMSO (300 μL) was placed under an argon atmosphere. Below, DIPEA (101.4 μL, 0.601 mmol) was added. The reaction mixture was kept stirring at room temperature for 2 hours and the progress of the reaction was followed by LC/MS. The final product was purified using preparative RP-HPLC (C18 column, 0% B-50% B (25 min), buffer A: 0.1 TFA (aq), buffer B: 0.1 TFA (acetonitrile solution) , λ=280 nm). After removing ACN under vacuum, the purified fractions were lyophilized to give a pale yellow solid.
LC/MS (ESI) (m/z): (M + H ) ⁺ calcd. for C46H53F3N10O16, 1058.9, found, 1059.0 g/mol.

[Example 22]
Synthesis of folate-Lys-PEG4-aminooxy linker (4).

Hydrazine (13 μL, 0.42 mmol) was added to a solution of folate-N10(TFA)-Lys-PEG-phthalimide (30 mg, 0.028 mmol) in DMSO (1 mL). The pH during the reaction was maintained at pH 9.35. Deprotection of phthalimido and TFA groups was monitored by LC/MS. Folic acid-Lys-PEG4-aminooxy linker was purified using preparative reverse-phase HPLC (C18 column, 0% B to 50% B (25 min), Buffer A: 0.1 TFA (aq), Buffer B: 0.1 TFA (acetonitrile solution) λ=280 nm). After removing ACN under vacuum, the purified fractions were lyophilized to give a yellow solid.
¹ H NMR (DMSO- _d6 / _D2O ) δ 1.88 (m, 1H, Pep-H); 2.03 (m, 1H, Pep-H); 2.15 (t, J = 7.4, 2H); 2.28 (t 3.05 (m, 4H); 3.20-3.60 (m, xH); 3.94 (m, 1H, Lys-αH); 4.23 (m, 1H, Glu-αH); 4.48 (s, 6.64 (d, J = 8.8 Hz, 2H, Ptc-Ar-H); 7.64 (d, J = 8.8 Hz, 2H, Ptc-Ar-H); 8.63 (s, 1H, Ptc-Ar-H); -Ar-H)
LC/MS (ESI) (m/z): (M + H) + calcd. for C36H52N10O13, 832.9 g/mol, found, 833.0 g/mol.

[Example 23]
Conjugation to folic acid-anti-CD3.

A solution of anti-CD3 Fab with pAcPhe (10 mg, 0.21 μmol) in PBS (pH 7.4, 1 mL) was buffer exchanged with 50 mM sodium acetate (pH 4.0, 900 μL). After adding 1 M acetic acid hydrazide (100 μL), folate-Lys-PEG4-aminooxy linker (1.7 mg, 2.10 μmol) was added to the reaction mixture with shaking (50 rpm) at 28° C. for 48 hours. incubated. The progress of the reaction was monitored by RP-HPLC (Zorbax 300SB C3 column, 4.6x150mm; Agilent). Complexes were eluted with a linear gradient from 30% B to 90% B (A: water and 0.1% TFA, B: acetonitrile and 0.1% TFA). Using an Agilent 1100 series HPLC system and Chemstation software, the percentage of Fab conjugated with folic acid-linker was analyzed and quantified. The reaction was completed within 48 hours with a conversion efficiency of over 95%. The final complex was purified using a cation exchange column (SP 650S, Tosoh Biosciences), buffer exchanged with PBS and stored at 4°C until use.
Expected MS: 48539.64 Da, Observed MS: 48538.89 Da.

[Example 24]
Study of efficacy in vitro.

Prior to use in the assay, target cells KB, OVCAR-3, SKOV-3 and OV-90 cells were incubated with folate-free RPMI-1640 (10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin) was maintained for 5 or more passages. Jurkat and A549 cells were maintained in RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were grown in monolayers at 37° C. in a 5% carbon dioxide, 95% humidity atmosphere.

To express FR, cells were incubated for 1 hour at 4° C. with anti-FR-PE antibody (blue) and IgG1 isotype-PE control (red), both from R&D systems. Binding was assessed by flow cytometry. KB cells or A549 cells were seeded in T75 flasks and allowed to form monolayers for 48 hours. After trypsinization, cells were transferred to centrifuge tubes (1×10 ⁶ cells per tube) and centrifuged. The medium was replaced with fresh medium with increasing concentrations of folic acid-FITC and incubated at 4°C for 30 minutes. After rinsing with fresh medium (2 x 1.0 mL) and PBS (1 x 1.0 mL), cells were resuspended in PBS (1.0 mL). Cell-bound fluorescence was then analyzed by flow cytometer (30,000 cells per sample). KB cells were plated in T75 flasks and allowed to form monolayers for 48 hours. After digestion with trypsin, the detached cells were transferred to centrifuge tubes (1×10 ⁶ cells per tube) and centrifuged. Spent medium in each tube was replaced with fresh medium (0.5 mL). This new medium contained 100 nM FA-FITC and increasing concentrations of anti-CD3 Fab-folate (0.1 nM to 0.8 nM). After 30 min incubation at 4° C., cells were rinsed with culture medium (2×1.0 mL) and PBS (1×1.0 mL) to remove any unbound radioactivity. Cells were then resuspended in PBS (0.5 mL) and cell-associated fluorescence was measured with a flow cytometer.

Human peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat (San Diego Blood Bank) by Ficoll Hypaque Plus (GE Heathcare) gradient centrifuge. When sufficient cells were not obtained from a single donor, multiple donors were used. However, for both in vitro and in vivo use, they were used separately from each other (ie, not mixed). PBMC were activated with aCD3/aCD28 supramagnetic beads (Life Technologies) in the presence of rhIL-2 (100 ng/mL) and GMCSF (100 ng/mL).

For cytotoxicity assays, target cells were detached from flasks with Accutase and plated in U-bottom 96-well plates. Activated PBMC (effector cells) or non-activated PBMC (effector cells) were added to target the cells at a specific (target cell: effector cell) ratio. Anti-CD3 folic acid was diluted in complete culture medium and added to the co-medium, briefly centrifuged, and the plates incubated at 37° C. in 5% CO ₂ for 24 hours. Regarding cytotoxicity due to LDH release, LDH was measured using a Promega kit according to the manufacturer's instructions. For cytotoxicity by flow cytometry, cells were labeled with DIO (stains the cell membrane green) and propidium iodide was added at a final concentration of 1 μg/mL ⁹ . Regarding cytotoxicity by ATP content, after 24 hours, PBMCs were washed away and ATP content of KB cells was measured using Cell Titer Glo (Promega).

[Example 25]
Pharmacokinetic studies.

Anti-CD3 Fab-folate and unconjugated CD3 Fab were administered to Sprague-Dawley rats (Charles River Laboratories). Dosing was a single bolus injection intravenously. Blood was collected at regular intervals and anti-CD3 Fab-folate in rat serum was quantified by ELISA. Time vs. serum concentration was used as an indicator of the pharmacokinetic properties of the conjugate.

[Example 26]
Study of effects in vivo.

6-8 week old female NOD-SCID mice were purchased from TSRI animal facilities or The Jackson Laboratory. Prior to the study, these mice were maintained on a folate-deficient, gamma-irradiated special diet (Teklad) for more than two weeks. Animals were housed 5 per cage, free of pathogens in a barrier system, and kept in covered shelves. Food and autoclaved tap water were available ad libitum. Individual mice were distinguished by tail base tattoos or ear piercings. All animal handling was approved by The Scripps Research Institute Animal Care and Ambrx, Inc. Animal Care and Use Committee. In addition, the handling of animals conformed to national and international guidelines for the humane treatment of animals.

Tumor cells (5×10 ⁵ KB cells per mouse) were mixed with activated PBMC or non-activated PMBC at a specific (target cell: effector cell) ratio. A 200 μL subcutaneous injection of this mixture was implanted into the left flank. Animals were treated with 1.5 mg/kg anti-CD3 Fab-folic acid or PBS intravenously once daily (total of 10 injections) (black arrows). The treatment began on the same day as tumor cell implantation, with an occasional 2-day withdrawal period to accommodate weekends. Tumor growth was monitored twice a week by measuring length in two perpendicular directions. Tumor volume was estimated by the following formula: V=(L×W×W)/2, where V is the volume, W is the shortest diameter, and L is the longest diameter. Body weight was measured by placing the mouse on an electronic balance. The experiment was terminated when the average tumor size of the group reached approximately 1,000 mm ³ or when it reached 35 days after the start of administration, whichever came first.

For immunohistochemical analysis, animals were killed by isoflurane and tumors and various organs were excised and fixed overnight in formalin-zinc. Tissues were embedded in paraffin and sectioned for staining. Hematoxylin and eosin (H&E) staining was performed according to a conventional method. Slides were scanned with a Leica scanner.

It is to be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or alterations that take into account these examples and embodiments will be suggested to those skilled in the art. sea bream. And it is to be understood that such modifications or variations are also included within the spirit and scope of the present application and within the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes.

The present invention can also be configured as follows.
<1> An anti-CD3 antibody having at least one of SEQ ID NOS: 1-15.
<2> The anti-CD3 antibody according to <1>, having two of SEQ ID NOs: 1 to 15.
<3> A bispecific binding molecule having (i) a first binding domain and (ii) a second binding domain, wherein the second binding domain consists of SEQ ID NOS: 1-15 A bispecific binding molecule selected from the group.
<4> A construct that has an amino acid sequence shown in one or more of SEQ ID NOs: 1 to 15, has cytotoxic activity and specifically binds to CD3.
<5> (a) a VH domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3, and (b) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6 An anti-CD3 antibody having a binding domain with a VL domain.
<6> The anti-CD3 antibody according to any one of <1> to <5>, wherein the antibody has one or more post-translational modifications.
<7> The anti-CD3 antibody according to any one of <1> to <5>, wherein the antibody is linked to a linker, polymer, or molecule having biological activity.
<8> The anti-CD3 antibody of <7>, wherein the biologically active molecule is folic acid.
<9> The anti-CD3 antibody according to any one of <1> to <5>, wherein the antibody is linked to a bifunctional polymer, bifunctional linker, or one or more antibody polypeptides.

Claims (12)

(i) a first binding site for a first antigen or epitope;
(ii) a second binding site for a second antigen or epitope that is different from said first antigen or epitope;
A bispecific anti-CD3 binding molecule having
The second binding site is
(a) a VH domain having the amino acid sequence of SEQ ID NO:3;
(b) a VL domain having the amino acid sequence of SEQ ID NO:6;
A bispecific anti-CD3 binding molecule having 2. The bispecific anti-CD3 binding molecule of claim 1, wherein said bispecific anti-CD3 binding molecule has one or more non-naturally encoded amino acids. 3. The bispecific anti-CD3 binding molecule of claim 2, wherein said non-naturally encoded amino acid is p-propargyloxyphenylalanine or p-azido-L-phenylalanine. 4. The bispecific anti-CD3 binding molecule of claim 2 or 3, wherein said non-naturally encoded amino acid is linked to a water soluble polymer. 5. The bispecific anti-CD3 binding molecule of Claim 4, wherein said water-soluble polymer comprises a polyethylene glycol moiety. The bispecific anti-CD3 binding molecule of any one of claims 1-5, wherein said bispecific anti-CD3 binding molecule has one or more post-translational modifications. 7. The bispecific anti-CD3 binding molecule of any one of claims 1-6, wherein said bispecific anti-CD3 binding molecule is linked to a linker, polymer or biologically active molecule. 8. The bispecific anti-CD3 binding molecule of claim 7 , wherein said biologically active molecule is folic acid. A therapeutic pharmaceutical composition comprising a bispecific anti-CD3 binding molecule according to any one of claims 1-8 . 10. A pharmaceutical composition according to claim 9 , comprising a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition comprising a bispecific anti-CD3 binding molecule according to any one of claims 1-8 . 12. A pharmaceutical composition according to claim 11 , comprising a pharmaceutically acceptable carrier or excipient.

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